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Advance in the toxic effects of petroleum water accommodated fraction on marine plankton Zhibing Jiang, Yijun Huang, Xiaoqun Xu, Yibo Liao, Lu Shou, Jingjing Liu, Quanzhen Chen, Jiangning Zeng Laboratory of Marine Ecosystem Biogeochemistry, Second Institute of Oceanography, SOA, Hangzhou 310012, China Abstract: Recently, the impact of petroleum pollution on marine plankton has been complemented by a great concern. This review summarizes the reports about toxic effects of oil water accommodated fraction (WAF) on marine phytoplankton, zooplankton and early life stage of animal. For the oil WAF, toxicants are mainly composed of the aromatic hydrocarbons, such as the benzene hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) with 2–5 rings. The oil WAF, especially the PAHs, can be accumulated in plankton due to their great lipophilic abilities, and thus elicites various deleterious effects. Toxicological tests show that marine plankton is very sensitive to the petroleum WAF, as the order of median effective/lethal concentration is merely μg/L or mg/L. There are species and developmental stages differences of plankton tolerance to petroleum WAF, and the toxicity of different oil WAF is various. Generally, its toxicity enhances with increasing carbonic chain length and benzene ring number. Many studies on the acute and sub-acute toxic effects of oil WAF have been done, however few researches on its chronic toxic effects has been carried out till now. Besides, most reports focused on the levels from molecule to individual, though very little work of petroleum toxic effects has ever been performed on the marine plankton population or community levels. Therefore, it is necessary to continue these studies in future. Key words: petroleum pollution; water accommodated fractions; polycyclic aromatic hydrocarbons (PAHs); phytoplankton; zooplankton; early life stage 1. Introduciton With the rapid economic development and energy demand, petroleum import amount is growing dramatically in China. Marine petroleum transportation and harbor throughput increase year after year, causing the frequency oil spill accident. Besides, oil pollution discharge from ship and crude oil exploration gradually increase. So the risks of marine oil pollution become more and more serious [1]. The import amount of crude oil was about 200 million in China in 2008 according to the statistics, above 90% of which was transported by sea. The realization of China’s Petroleum Strategy Reservation Plan and the increase of crude oil demand in future will accelerate the oil transportation amount by sea. Therefore, the risks of marine oil pollution will continuously climbing in China, and the problem of marine ecological safety can not be optimistic. China marine environmental quality communique in 2008 [2] showed that oil still was the one of three main pollutant (the other two were nitrogen and phosphorus) in coastal areas, especially in some Corresponding author. Email address: [email protected] (J. Zeng). important half blocked bay with less water exchange. This ecological risk due to the long term accumulation of oil pollution is severe and can not be ignored [3]. Marine phytoplankton, as the most important primary productivity, can offer food to zooplankton and larvae and juvenile fish [4]. Zooplankton can influence or control the primary productivity by top-down effects [5] in return, and its population dynamic change can influence the biomass of other marine animals like fish by bottom-up effects [6]. Once the community of plankton changed by the effects of oil pollution, the structure, stability and function of marine ecological system can be changed as well [7]. It is necessary and crucial to study the impact of oil pollution to marine plankton in view of the serious oil pollution of coastal areas in China and the ecological system function and status of marine plankton. Current researches focus on the impact of crude oil water accommodated fraction (WAF) to marine phytoplankton [8–11], zooplankton [12,13], and animals in early life stages [14,15]. This paper summarized the research results about the influence of oil WAF on marine plankton both at home and abroad, and prospected the study points in future, to further promote the quantified evaluation of the damage by oil pollution to marine ecology. 1. The composition and main toxic substances of marine petroleum WAF Petroleum (crude oil) is a complex mixture that consisted of hydrocarbon (including alkanes, cycloalkanes and aromatic hydrocarbons) and non-hydrocarbon (including resin and asphalt). A series process of physics, chemistry and biology diversifications happen after crude oil entering the ocean, including spread, evaporation, dissolution, emulsification, disperse, absorption, sedimentation, biological decomposition and photo-oxidation [16]. These oil substances will be partially physically transferred and biologically decomposed, and the rest will dissolve in the seawater. Different kinds of crude and refined oil have different compositions, water solubilities (generally 200×10−6 mg/L [17]), and WAF components. However, WAF is mainly constitute of BTEX (the general term of benzene, toluene, ethylbenzene and xylene), alkylation of benzene homologues, polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbon and some unresolved complex mixtures (UCMs) through chromatogram [18]. The mainly toxic substances are some aromatic hydrocarbons, such as BTEX and PAHs. The PAHs in WAF are mostly 2–5 rings [18] and the PAHs with 6 rings are little, because the rest PAHs with more rings can not dissolve in water for their highly lipophilic ability (high logKow value). Heterocyclic compounds of N, P, S in petroleum also has contribution on the toxicity, such as thiophene and its alkylated homologues, quinoline, acridine and other components with high water solubility and toxicity [19]. And these heterocyclic compounds can be gradually accumulated in the process of oil weathering [20]. But, generally speaking, the heterocyclic compounds are much less than BTEX and PAHs in oil [21], so they are relatively lowly toxic in the total toxicity. The resin and asphalt are hard for the living organism absorption, due to their high molecular weight (700–1000 of resin and 1000–10000 of asphalt [22]). So they are also less toxic to marine plankton. The PHAs with > 2 rings mainly have chronic toxic effects to the environmental damage and organism hurt [23,24], for they are difficultly decomposed [25]. The BTEX and the naphthalene with 2 rings and its homologues have acute toxic effects, due to their high concentration and water solubility and easily volatile that can not stay long in the water [25]. 2. The toxic effects of oil WAF to marine phytoplankton Different kinds of oil WAF have different toxicity to phytoplankton, and generally, the longer the carbon chain is, the more benzene ring has, and the more toxic the oil is. The order of toxicity (Table 1) to phytoplankton usually is ortho xylenes > toluene [10], benzo (a) pyrene (5 rings) > pyrene and fluoranthene (4 rings) > anthracene and the phenanthrene (3 rings) > naphthalene (2 rings) > BTEX (1 ring) [10,26–29], heavy oil > light oil [30,31], aromatic hydrocarbons > alkanes [31]. Besides, different phytoplankton species has different tolerance to oil WAF toxicity, representing different median effective concentration (EC50) of growth (Table1). Phytoplankton community happen abnormal succession under the stress of oil pollution that the dominant degree of less tolerant species gradually decreases, even vanishes, while more tolerant species gradually become the dominant ones [8,9,31–33]. The field investigation after oil spill accident of “TASMAN SEA” tanker showed that diversity of phytoplankton community decreased around the pollution area, and species number and cell density reduced to 1/2 of pre-accident, especially diatom, the dominant species in population, its species number climbed down from 40 to 18. Contrarily, the species number of dinoflagellates did not change [1]. Table 1 EC50 of growth inhibition for different phytoplankton species under different toxicants exposure Species Toxicant 72h-EC50 Pheodactylum tricornutum Phenanthrene 154 ± 3.1 μg/L Anthracene 123 ± 5.5 μg/L Fluoranthene 103 ± 9.1 μg/L Pyrene 119± 1.2 μg/L Phenanthrene 47 ± 5.5 μg/L Anthracene 39 ± 2.4 μg/L Fluoranthene 18 ± 2.9 μg/L Pyrene 24 ± 2.0 μg/L Fluoranthene 1031 μg/L Pyrene 260.3 μg/L Benzo(a)pyrene 55.24 μg/L Cyclotella caspia Fluoranthene 0.20 mg/La [29] Chlorella uvlgaris 0# Fuel oil WAF 12.11 mg/L [30] 0# marine fuel oil WAF 12.22 mg/L Heavy fuel oil WAF 18.73 mg/L BTEX 62.91% WAF a mixture of aromatics with C9-C11 28.59% WAF a Raw naphtha 27.84% WAF a Light naphtha 28.59% WAF a Heavy naphtha 4.91% WAF a mixture of aromatic hydrocarbons of C9 4.79% WAF a mixture of C6-C8 hydrocarbons, paraffin and isoparaffin 19.92% WAF a Toluene 34.10–114.00mg/L Naphthalene 3.90–7.30 mg/L Ortho xylenes 1.69–3.03 mg/L Skeletonema costatum Thalassiosira pseudonana Tetraselmis chuii Zooxanthella croadriz tica, P. tricornutum, Nitzschia closterium minutissima, S. costatum, C. uvlgaris, Reference [26] [27] [28] [31] [10] Platymonas subcordiformis Phenanthrene 0.60–1.92 mg/L Unmarked letter indicated 72h-EC50; a: exposure duration for 24 h Both laboratory researches and field investigations showed that low concentration of WAF in seawater had less influence on phytoplankton, even could promote its growth [9,34,35]. The stimulative effects were related to the bacteria in water and algae itself [34,35]. The bacteria would decompose the oil WAF to the algae utilized carbon. And some species of phytoplankton, such as Chlamydomonas, Chlorella, Navicula, Nitzschia and Cyclotella, could decompose WAF by themselves and realize the biological transformation [29,36,37], consequently achieve the biological restoration of oil pollution area. But if the oil concentration was too high, it could restrain the algae growth [11,38,39]. Therefore, it is worthy of being discussed that how much the concentration was low or high need the experiments to judge. Whether the degrees of oil pollution in China can influence coastal phytoplankton community, and if the influence exits, how much the influence is in different seasons, and whether such influence can cause the decreasing species diversity, dominance change, or community abnormal succession. Oil WAF accumulated in the phytoplankton cells [40], suppressed their photosynthesis by reducing the primary photochemical yield, electron transport capacity, the activity of release oxygen center and photosystem II. Meanwhile respiration of phytoplankton enhanced, compelling the increase of energy expenditures [41]. WAF also could block the absorption of CO2 and nutrients, leading to the decreasing chlorophyll a and reduce of primary productivity [33,42]. Oil WAF would destroy the cell structure and membrane system of microalgae [43], disturb the operation of anti-oxidation defence system [41,44], stop the synthesis of nucleic acid and protein [45], even induce the cell abnormality [29,46] and gene mutation [28]. Researches showed that oil WAF would damage DNA structure or cause the DNA adduct formation of microalgae cells[28,47], prevent the replication of DNA, thus cells could not divide or proliferate, leading to the augment of cell size [33,48]. But Wang et al. [49] found that oil WAF did not make the granularity of Chaetoceros curvisetus increase, and the reasons needed the further study. 3 The toxic effects of oil WAF to marine zooplankton Generally, the toxicity of oil WAF enhances with the increment of carbon chain and benzene ring (Table 2 and 3). The order of toxicity to the same species of zooplankton were dimethylnaphthalene > methylnaphthalene > naphthalene [50,57], pyrene and fluoranthene > anthracene and philippines > naphthalene [51,54], and kerosene > gasoline > diesel oil > crude oil [15]. But Wang et al. [53] found that the toxicity of anthracene and philippines was stronger than pyrene and fluoranthene, as artemia nauplii being the study object. Besides, the tolerance of zooplankton to oil WAF has the species differences as well as growth and development stage differences. For example, the median lethal concentration (LC50) of naphthalene and its homologue to Oithona davisae [57] was greatly higher than to Paracartia grani [50], the LC50 of fluoranthene to nauplius, adult and egg of Tisbe battagliai were 68.3, 101.1 and 86.2 μg/L, respectively [52]. On the aspect of acute lethal, oil WAF can cause the activity decrease and non-polarity narcotic coma, even to death, by affecting the lipid membrane fluidity of zooplankton, and interrupting operation of physiological and biochemical system[58–60]. On the other aspect of sub-acute and chronic lethal, oil WAF will disturb zooplankton feeding, spawning, hatching, growth, development and behavior (Table 2), consequently bring out the long negative effect to the population activity and continuity. Bejiarano et al. [62] took the experiments that Amphiascus tenuiremis was exposed in South Louisiana crude oil for three generations, and they found that the population exhibited abnormal growth and development, and the reproduction and population yield both greatly reduced. Currently, there are few researches about the chronic toxicity of oil WAF to zooplankton and its effect to the population and community. So assessment of population damage caused by oil pollution was lack of basis. The toxicity experiments are quite needed to study the effects to the full lifecycle, and the mesocosm experiment are highly demanded to research the response of population and community level to oil pollution. It is not difficult to measure the toxicity of whole lifecycle of zooplankton, for their short generations and being easily controlled. Table 2 EC50 of feeding, spawn and hatch for different copepod species under different toxicants exposure Species Toxicant EC50 EC50 Feeding rate Paracartia grani 1264 μg/L Naphthalene 1,2-dimethylnaphthalene Acartia tonsa μg/L a ― Fluoranthene Tisbe battagliai 146 a EC50 Clutch size 2096 254 Hatching rate μg/L a ― μg/L a 87.58 Reference [50] ― μg/L b 77.87 μg/L b μg/Lb Phenanthrene ― 253.98 Pyrene ― 61.89 μg/L b 59.67 μg/L b Fluoranthene 34.0 μg/L c 66.9 μg/L d 63.4 μg/L e 180.37 [51] μg/Lb [52] A: 24h-EC50; b: 48h-EC50; c: Exposure time = 2–4 d; d: 6d-EC50; e: Exposure time >4 d Table 3 LC50 of different zooplankton species under different toxicants exposure Species Life stage Toxicant 48h-LC50* A. tonsa Adult Fluoranthene 120.14 μg/L Phenanthrene 105.87 μg/L Pyrene >129.45 μg/L Naphthalene 2523 μg/L a P. grani T. battagliai Artemia salina Mysidopsis bahia M. bahia Adult Nauplii 1,2-dimethylnaphthalene 161 Fluoranthene 68.3 μg/L b 101.1 μg/L Egg 86.2 μg/L c Larvae (24–48 h) ― Phenanthrene [51] [50] μg/L a Adult II–III instar nauplii Reference [52] c 1320.7±19.8 μg/L a Anthracene 1009.5±37.5 Fluoranthene 1430.5±13.4 μg/L a Pyrene 1770.6±213.1 μg/L a Anthracene, Fluoranthene, and Pyrene 535, 63.8, and 24.8 μg/L 2# 548 μg/L Fuel oil (TPAHs) WAF [53] μg/L a Arabian Light Crude oil (TPAHs) WAF 139 μg/L Prudhoe Bay crude oil (TPAHs) WAF 157 μg/L Weathered Guadalupe oil WAF (TPHs) 0.92 (0.71–1.14) mg/L d [54] [55] M. bahia <1d Ampelisca abdita Juvenile Oithona davisae Nauplii Adult Calanus sinicus ― Fluoranthene [56] 67 (59–76) μg/L e Naphthalene 4422 (3942–4961) μg/L a 1,2-Dimethylnaphthalene 771 (759–784) μg/L Naphthalene >10 mg/L a 1,2-Dimethylnaphthalene 1346 (1047–1732) μg/L a Daqin crude oil WAF 19.8 mg/L Diesel oil WAF 15.8 mg/L 70# 6.1 mg/L Gasoline WAF Kerosene WAF 31 (22–41) μg/L e [57] a [15] 3.5 mg/L Unmarked letter indicated 48h-LC50; a: 24h-LC50; b: Exposure time > 4 d; c: 6 d-LC50; d: 7d-LC50; e: 96h-LC50; WAF: Water accommodated fraction; TPAHs: Total polycyclic aromatic hydrocarbons; TPHs: Total petroleum hydrocarbons Traditionally considering, PAHs with more benzene rings and higher molecular weight are not much in the water, due to the low solubility [18], and usually sinking to the seafloor with organic matters. However, lately researches showed that PAHs still stay in the seawater for a relatively high concentration [32,63]. Oil WAF mostly are strongly lipophilic [58], so the organic compounds (especially PAHs) of WAF can be accumulated in the body of marine zooplankton with high body fat [64] by utilizing the routes of feeding and surface contact [65,66], which is definitely no good to the zooplankton. Even if exposed in the low concentration of oil WAF, zooplankton also will behave abnormally that feeding rate is decreasing [13,50,52] while oxygen consumption is increasing [67]. Then the content of carbohydrates, protein and fat, and the activity of electronic energy transfer system are affected, and the balance of energy budget is broken [68], thus leading to the abnormal growth and development, and the decrease of growth and breeding rate [51,62,69]. 4. The toxic effects of oil WAF to marine fauna in early life stage The early life stage of marine animals refers to the animals’ development from the fertilized egg to planktonic larvae. The fertilized eggs and planktonic larvae of marine animals, for their special ecological status and economic function, are classified as another group of zooplankton in this paper. Large quantities of oil WAF can be accumulated in the yolk of fertilized eggs [59,70,71] for its strong lipophilic ability [58]. Thus, there are high concentrations of PAHs in the original embryonic development stages of marine fauna. When the animals began to develop and the embryo assimilated the necessary nutrients from the yolk sac, PAHs began to disperse inevitably into the embryo, affecting its physiological and biochemical processes of early development [72], then affecting embryo development, and organ differentiation and formation [70,73,74]. Since early life stage of marine fauna involved most of life processes, including cleavage, growth, basic metabolism and synthesis, organ differentiation, formation and development etc., it was more sensitive to pollution in the early life stage than any other life stages. Therefore, it was considered as the most important and crucial stages of life. Base on it, the fertilized eggs and larvae are widely used in marine toxicity test (Table 4 and 5). Table 4 and 5 showed that most kinds of fertilized eggs, embryos, and larvae of marine fauna was sensitive to the oil WAF, because their unit of LC50 and EC50 were only μg/L or mg/L. Different origins, efflorescence degrees [70] and kinds [54,8,77] of oils were different in their toxicities. But generally, the toxicity of oil WAF to the embryos and larvae enhances with the increasing of carbon chain and benzene rings. Also the tolerances of embryos and larvae to oil WAF were different among species and developmental stages [8,54,77]. Besides the lethal effects, more serious circumstances was that oil WAF could also cause the conformation deformities and developmental abnormalities to the early life stages of marine animals. Take the larval fish for example [70], oil WAF would cause the reduced hatching rate, developmental malformations (such as spinal curvature and yolk sac edema), membrane phase delay, decrease or loss of feeding and swimming ability, growth inhibition, interference with nervous system function, decreased immunity, as well as increased risk of predation, etc.; As in the case of shellfish [54,73,74], it could result in reduce or deformity of D-veliger, failure of larval metamorphosis, inhibition of growth and development, etc. (Table 5). Carls et al. [70] studies showed that > 0.4 μg/L of TPAH concentrations could multiple the occurrence frequency of yolk sac edema, and > 0.7 μg/L of it could lead to reduced of fish body length, and rapidly increased of yolk sac edema and spinal deformities. These sub-acute or chronic toxic effects were bound to influence the maintenance and continuance of marine animal populations. Table 4 LC50 of different marine fauna (Early life stage) species under different toxicants exposure Species Life stage Toxicant 96h-LC50 Reference Palaemonetes pugio Larvae (< 48 h) Fluoranthene 2.53 (2.14–2.58) μg/L [71] Benzo(a)pyrene 1.02 (0.83–1.26) μg/L Melanotaenia fluviatilis Egg Bass Strait crude oil (TPHs) WAF 1.28 (1.0–1.6) mg/L Naphthalene 0.51 (0.4–0.7) mg/L [75] Homarus americanus Larvae Fluoranthene 317 (166–378) μg/L Clupea Pallasi Egg Less-weathered Alaska North Slope crude oil WAF 53.3 ± 3.6 μg/L Anthracene, Fluoranthene, and Pyrene 4260, 58.8, and >11900 μg/L Mulinia lateralis egg Sparus macrocephalus Pagrasomus major larvae 0# (15–20 mm) 20# Diesel oilWAF Diesel oil WAF [14] 3.02 (1.95–4.47) mg/L 3.55 (2.51–3.98) mg/L larvae 0# 0.71 (0.54–1.07) mg/L (18–22 mm) 20# Diesel oil WAF Diesel oil WAF 3.16 (2.19–4.57) mg/L South China Sea crude oil WAF 5.89 (4.07–8.51) mg/L Shengli crude oil WAF 6.4 (4.9–8.5) mg/L a Paralichthys olivaceus 13.7 (12.3–15.2) mg/L S. macrocephalus 10.7 (7.5–15.1) mg/L a Unmarked [54] a 0.28 (0.23–0.35) mg/L South China Sea crude oil WAF larvae (3–5 d) [70] a >13300, 3310, and >9454 μg/L Larvae and juvenile Penaeus monodon [56] [77] a letter indicated 96h-LC50; a: 48h-LC50; WAF: Water accommodated fraction; TPHs: Total petroleum hydrocarbons Table 5 EC50 of different marine fauna (Early life stage) species under different toxicants exposure Species Life stage Toxicants Index 48h-EC50 References Mytilus Egg–D-veliger Naphthalene Percentage of D-veliger 9.92 (9.36–11.77) mg/L [73] galloprovincialis Phenanthrene 224.21 (173.42–298.89) μg/L Ciona intestinalis Paracentrotus Egg–Tadpole larvae Egg–Pluteus lividus M. Percentage of tadpole 4.28 mg/L Phenanthrene larvae >427.75 μg/L Naphthalene Larval growth 558.82 (483.20–645.98) μg/L 418.16 (366.64–512.23) μg/L Phenanthrene Eggs–D-veliger galloprovincialis P. lividus Naphthalene Prestige fuel oil WAF Percentage of D-veliger Marine fuel oil WAF Egg–Larvae ― Prestige fuel oil WAF Weathered Guadalupe [74] > 100% WAF Larval length Marine fuel oil WAF M. bahia 17 (16.9–17)% WAF 11 (9–14)% WAF 58 (38–88)% WAF oil WAF Dry weights 0.21 mg/L a Spinal abnormality 33.5±3.1 / 3.60±0.55 μg/L b Yolk sac edema 19.6±1.6 / 0.77±0.16 μg/L Small jaw 22.3±1.4 / 1.00±0.21 μg/L b Effective swimmers 18.4±1.1 / 2.44±0.29 μg/L b Dry weight >11900, 900, and >9454 μg/L c [55] (TPHs) C. pallasi Egg–Larvae Less-/more-weathered Alaska North Slope crude oil (TPAHs) Mulinia lateralis Juvenile Anthracene, Fluoranthene, and Pyrene 2# Fuel oil (TPAHs) WAF 806 [70] b [54] μg/L c 118 μg/L c Arabian Light Crude oil (TPAHs) WAF >2540 μg/L d Prudhoe Bay Crude oil (TPAHs) WAF Hemicentrotus Egg–Pluteus Marine heavy fuel oil WAF 0# Diesel pulcherrimus Larval growth oilWAF 2.71 (2.21–3.32) mg/L c 1.87 (1.69–2.06) mg/L Unmarked letter indicated 48h-EC50; a: 7d-EC50; b: 16d-EC50; c: 96h-EC50; d: 72h-EC50; WAF: Water accommodated fraction; TPAHs: Total polycyclic aromatic hydrocarbons; TPHs: Total petroleum hydrocarbons 5. The toxicity and detoxification mechanism of oil WAF to marine plankton Quantitative structure–activity relationship (QSAR) studies show that the toxicity of organic compounds depends primarily on the accumulation capacity in organisms and the ability to interact with the receptors [79]. Oil WAF toxicity mechanism to plankton approximately is that: most WAF are strongly lipophilic, and the fat contents are usually high in the body of marine plankton [63]. Thus, quantities of these organic compounds (especially PAHs) are accumulated in marine plankton [22,40,66], affecting the physiology, biochemistry, behavior, reproduction, growth, development and survival. PAHs with 2–3 rings showed strongly acute toxicity, but generally no carcinogenic effect, while PAHs with 4–5 rings showed strongly effects of carcinogenicity, teratogeny and genetic mutation, not the acute toxicity [80]. Currently, the theories on the carcinogenic mechanism of PAHs are mainly K-region theory, Bay-region theory, and Di-region theory etc., but all of which have not yet been completely clarified [80]. The toxicity mechanisms of BTEX and PAHs to plankton are non-acute anesthesia, adduct formation, reactive oxygen free radicals, and endocrine disruption [69]. The different components have different toxic mechanisms. For instance, BTEX and the naphthalene with 2 rings mostly operate by the anesthesia [59] or Lipid-partitioning mechanisms [81], while PAHs with 3–5 rings work mainly by the metabolism-driven approach [70], including the generation of reactive oxygen species (ROS) from cells under the oxidative stress and the toxic intermediates or metabolites induced by system of cytochrome P-450 monooxygenase (also known as Mix-function oxidase, d [78] MFO) [82,83]. At present, the knowledge about toxicities of PAHs with small ring and multiring still have uncertainties [84,85], existing additive effects [60] as well as antagonistic and synergistic effects [69]. In general, BTEX and PAHs into the organisms are formed into the aromatic rings oxide and ROS by MFO catalyzation, and then come into being aromatic hydrocarbon dihydrodiol diol derivatives by hydrolysis of hydrase, which can form the carbocation with electrophilic [86,87], binding to guanine N-2 of DNA molecule forming. Then DNA adducts are formed by DNA alkylation, leading to the change of the DNA base structure, consequently resulting in the genetic code change, causing mutations, and finally inducing the cancer [80]. At the mean while, ROS may damage proteins, lipids, DNA and other biological macromolecules [88], then bringing about the distortion. The detoxification processes of oil WAF in vivo are constituted of Phase I reaction and Phase II reaction. Phase I reaction: the lipophilic oil WAF catalyzed by MFO is transformed into more polar material by the introduction of the corresponding reactive bases through a series of oxidation, hydrolysis and reduction reactions, in order to create the conditions for Phase II reaction. Phase II reaction is to combine the exogenous substances (oil WAF) and its metabolic intermediates (like aromatic ring oxide) with some endogenous substances, which is catalyzed by glutathione S-transferase. So oil WAF and its intermediates are more hydrophilic and easily eliminated from the body. However, PAHs with 4–5 rings, originally as an indirect carcinogen, can be the metabolic intermediates (PAH epoxide) with carcinogenic effect and the ultimate carcinogen (dihydrodiol diol epoxy PAH) in this process [80]. It is obvious that the toxicity of oil WAF can be reduced or eliminated by the bio-transformation on the one hand, but may increase on the other hand. PAHs can be eliminated out of the body by the fast and effective metabolism through the active MFO system in vertebrates, but accumulated in the body of invertebrates lack of MFO system activity [85]. Marine plankton, except the larval fish as vertebrates, most are invertebrates. Moreover, larval fish are not fully developed. Therefore, the low concentration of PAHs can also bring about the damage to them [70]. 6. Problems and prospect The low concentration of oil WAF can lead to some species microalgae bloom, for the phytoplankton has short life cycle and rapid generation turnover. At the mean while, other consumers, such as the zooplankton with the longer generation alternation, have poor tolerance to oil pollution. So the biomass need a longer term to recover, resulting in the top-down effects weakening or loss of control [8,9,42]. In such mismatch effect of circumstances, once the nutrients and other physical and chemical factors are desirable and adequate, red tide may be triggered around the oil pollution area [89], such as the outbreak of Huanghua red tide in 1989 [90]. Therefore, we should take full account of the relationship between oil pollution and red tide outbreaks. It was reported that certain single toxicants of oil WAF were usually tested on plankton to value the biological toxicity, but the conclusions of this method were often different from the real situation, for the actual toxicities of oil, as a complex mixture of toxic ingredients with different proportion of each components [18], were far more complex than the single toxicant under the effects of mixed-toxic additive, antagonistic or synergies [23]. The researchers had disputes on the combined toxicity of mixed PAHs to plankton. So the complex mechanisms of toxicities are still needed for the further study, and the methods of QSAR and experimental ecology are necessarily utilized to verify the results. In addition, WAF toxicological test can also be applied for the more accurate assessment of petroleum pollution to plankton. At present, the toxicities of oil WAF on marine plankton mostly focused on the acute or sub-acute researches, few on the chronic ones. And most of these toxic studies emphasized on the levels of biochemical, molecular, sub-cells, cells, tissues, organs and individual, few on the aspects of populations or communities. Actually however, study the impact of toxins on the individual is to better study the population effects, for the primary objective of ecotoxicology is to ensure the populations durative and vigor in ecological communities [88]. Therefore, the impact of oil WAF to the maintenance and activity of marine plankton population are needed to be emphasized for the future study. Many works had been taken about the impact of oil pollution to marine plankton in global. However, few studies were found in China. And different species (populations) of plankton respond to oil pollution differently. Therefore, it is necessary to continuously carry out the toxicological response of plankton species, populations and communities to oil WAF in China's coastal areas, in order to provide the scientific basis for the quantitative evaluation of marine biological resources and ecological loss caused by the oil spill and oil pollution. The studies about oil WAF toxic effects on marine plankton mostly were carried out under the laboratory light conditions, not under the simulated sunlight so far. However, a large number of studies showed that the ultraviolet radiation from sun significantly enhanced the toxicity of oil WAF on plankton [26,47,71,74], which was called “phototoxicity”. Obviously, the results of laboratory experiments without the measurement of UV radiation to predict the oil WAF impact on marine life often would underestimate the toxic effects of oil pollution under the natural light and lead to the underassessment of ecological damage caused by oil spills and pollutions. Acknowledgements The project was financially supported by National Basic Research Program of China (No. 2010CB428903), Scientific Research Fund of Second Institute of Oceanography, SOA, China (JG0921, JT0806), National Marine Public Welfare Research Project of China (No. 200805069), National Special Fund of China (908-01-BC06, 908-02-04-02, 908-ZC-II-04), and SOA Special Fund of China (No. 2011914). References: [1] Gao Z H, Yang J Q, Wang P G, et al. Theory, method and case study of the ecological risk assessment of marine oil spill. Beijing: Ocean Press, 2007 [2] State Oceanic Administration. China marine environmental quality communique in 2008. 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