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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-11-02 - 2005-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Principles of method if other than guideline:
Based on a range-finding experiment which indicaated raapid metabolism, the uptake phase was scheduled for 56 hours, the depuration phase for 30 hours.
GLP compliance:
yes
Details on sampling:
- Sampling intervals/frequency for test organisms: Uptake = 1, 2, 4, 8, 12, 32,and 56 h;Depuration = 1, 4, 16 and 40 h
- Sampling intervals/frequency for water samples: Uptake= 0, 1, 2, 4, 8, 12, 32,and 56 h; Depuration = 1 h

During the uptake phase scheduled for 56 h, the fish were continuously exposed to the test substance. Thereafter the fish were transferred into new aquaria served with test substance-free dilution water further 30 h.  The duration of both, uptake and depuration phase, were fixed based on the results of a non-GLP pilot study. On each sampling time, samples of 4 fish per concentration were removed from the test vessels according to the sampling schedule (Table 1) and immediately rinsed in dilution water, blotted dry and killed. To avoid degradation of the test substance by  residual activity of esterases, the fish were either immediately frozen in liquid nitrogen or homogenized in organic solvent.  The fish were individually analyzed for the test item by GC-MS/MS. At each fish sampling time during the uptake phase, and 1 h after start  of the depuration phase, adequate amounts of water were analyzed per concentration. The water samples were analysed immediately after  sampling. 
Details on preparation of test solutions, spiked fish food or sediment:
Stock Solution: The stock solution was prepared two times a day under sterile conditions without solvent in a concentration close to the water solubility limit of ethylhexyl methacrylate. To prepare a stock solution of 2.5 mg/l, 25 mg  of the test item were mixed with 10 L of water in sterilized vessels and agitated at room temperature for at least 21 h. Then, 9 L of the aqueous phase (the saturated solution) were drained from the port at the bottom  of the mixing vessel into a sterilized stock solution bottle.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Specification: Danio rerio (Cyprinidae, Teleostei) Hamilton Buchanan
Size: juvenile fish, age 1.5 - 2 months (discrimination of sex not possible)
Source: laboratory bred
The fish were originally obtained from:
West Aquarium GmbH
37431 Bad Lauterberg
Loading: 0.3-0.4 g/L test vessel volume = < 0.1 g/L test solution*day)
Food: no feeding during the test
Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
56 h
Total depuration duration:
30 h
Test temperature:
24 +/- 1°C
Details on test conditions:
Fish exposure: The challenge of this study was to maintain the test concentration in water despite the rapid dissipation of the test item by diverse processes, i.e., degradation by bacteria, adsorption and volatilization and metabolism by fish. A non-GLP pre-study was performed to develop and optimize the approach resulting in maintaining a 5-fold exchange of test solutions at a low fish density (< 0.1 g/L*day).

Groups of 48 fish each were exposed to nominal concentrations which were  based on the results of the non-GLP pre-study and aimed to be 0.3 mg/L  and 0.06 mg/L 2-Ethylhexyl methacrylate in a flow-through system at a test temperature of 24 +/- 1°C. The concentrations of the test substance in the fish and in the water were determined throughout the different  phases of the test. An effect control group was kept under comparable conditions without adding test substance; the fish were adequately reduced and used for the determination of fat content.

All-glass aquaria containing 20 L of test solution were used as test vessels. A continuous flow of approximately 4.2 L/h test solution (uptake phase) or water (depuration phase) was maintained throughout the test using metering pump systems, resulting in a water exchange rate of 5 times per day.Observations were made throughout the test period on fish behaviour and mortalities. Temperature, pH and the oxygen concentrations in the test vessels were measured daily. The light/dark cycle was adjusted to 12/12  hours.
Nominal and measured concentrations:
0.3 mg/l and 0.06 mg/l (nominal)
Reference substance (positive control):
no
Details on estimation of bioconcentration:
Calculation of the uptake and depuration rate constants and the BCF: The uptake rate constant (k1), the depuration rate constant (k2), the  kinetic steady state bioconcentration factor (BCFk) were calculated by  linear and nonlinear regression functions using data for concentrations  of 2-Ethylhexylmethacrylate in whole fish measured in the extracts.  Calculations of means and ranges were done with Excel spreadsheets  (Microsoft Inc.) while linear and non-linear regressions were conducted  with the program SigmaStat 2.03 (SPSS Inc. 1997).

Calculation of the steady state BCF: The test substance is known to be taken up quickly due to the high  partition coefficient and to be rapidly metabolized, leading to a very  fast elimination. A non-GLP pre-study showed that the steady state can be  expected to be achieved within the first 8-12 hours. Two further sampling  dates after 32 h and 56 h were included to provide certainty about the  BCF. The BCFSS was calculated by dividing the mean of the values for the  2-Ethylhexyl methacrylate concentration in fish which represent the worst  case steady state by the mean measured relevant concentrations in the  water.   

Calculation of the depuration rate constants: The depuration rate constant (k2) was calculated using the measured  concentrations in fish during the depuration phase by applying a model  regarding fish as one compartment. The model assumes that the  concentration of the test substance in the fish (Cf) is decreasing  exponentially: Cf(t) = Cf(ti) * e(-k2*t) Cf(t):  concentration in fish at sampling time  in days (µg/Kg)  Cf(ti): steady state concentration in fish corresponding to the concentration at start of the depuration phase (= 100%) k2:  depuration rate constant k2 was calculated by linear regression applied to the ln-transformed  concentrations in fish.

Calculation of the uptake rate constant: The uptake rate constant k1 was calculated by a non-linear regression of  the ratios Cf/Cw against time during the uptake phase and using the  depuration rate constant fitted before. The fitted model assumes an  attenuation of uptake by simultaneous depuration, increasing with  increasing Cf up to an steady state between uptake and depuration. For the one compartment kinetics eq. 3 was fitted: Cf/Cw = k1/k2 * (1-e(-k2*t)) k1:  uptake rate constant Cf:  concentration in fish (µg/kg) Cw:  concentration in water (µg/L) k1 was calculated by non-linear regression using the k2 values obtained  in the depuration phase .

Calculation of kinetic BCFk:  The kinetic BCF for the one compartment model is given by  BCFk = k1/k2
Key result
Temp.:
ca. 24 °C
pH:
8.5
Type:
BCF
Value:
37 dimensionless
Calculation basis:
kinetic
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
1.5 h
Elimination:
yes
Parameter:
other: DT95
Depuration time (DT):
6 h
Details on kinetic parameters:
Uptake of 2-EHMA by fish and steady state BCF (BCFss): The steady state is reached at 1.170 mg/kg fish at 0.033 mg/L and 2.758  mg/kg fish at 0.082 mg/L. When calculating the BCFss by dividing the  conservative steady state concentrations in fish by the mean  concentration measured in water for the same sampling dates, the results  are BCFss = 35 at the low treatment concentration BCFss = 34 at the high treatment concentration

Depuration In the depuration phase concentrations in the fish were below LOQ after 16 h. Consequently, only the data for 0, 1 and 4 h were used to calculate the depuration rates. The depuration rates were calculated to be 0.51 and 0.47/h and thus very similar for the low and the high treatment groups, respectively, indicating a depuration half-life of less than 1.5 h and 95 % depuration within approximately 6 hours.

Determination of k1 and BCFk Uptake rates were calculated to be 19.2 and 17.4/h and thus very similar  for the low and the high treatment level, respectively. From the kinetic  rates the BCFk was calculated to be 37.5 and 37.4 for the low and high  treatment group, respectively.
Details on results:
No adverse effects or mortalities were observed during the study.

During the uptake phase, all concentrations in water and fish were above  the LOQ.

After equilibration of the test system, the nominal test concentrations estimated from the non-GLP pre-study of 0.3 and 0.06 mg/L were nearly met. After addition of the fish, the concentration in water in the low  treatment group, rapidly decreased to 50% of nominal during the first 2 h  of exposure. It remained sufficiently constant at a mean measured  concentration of 0.030 mg/L thereafter. In the high treatment, the  concentration in water rapidly decreased to 30% of nominal during the  first 4 h of exposure. It remained sufficiently constant at a mean  measured concentration of 0.082 mg/L thereafter. 
Reported statistics:
SQS 2000: Software for statistical quality assurance of analytical data.

Test item concentrations in water and fish, and ratios between both for  the low and high test concentrations

       time    c_water  [mg/L]   c_fish [mg/kg]    c_fish/
D       h       low    high       low    high      c_water*
0.0     0      0.066   0.378     0       0          0    0
0.04    1      0.049   0.181     1.146   4.607     20   16
0.08    2      0.035°  0.107     1.232°  5.482     29   38
0.17    4      0.028°  0.088     1.072°  3.452     34   35
0.33    8      0.032°  0.081°    1.064°  3.140°    33   39
0.5    12      0.038°  0.081°    1.310°  2.634°    34   33
1.33   32      0.027   0.084°    0.651   2.171°    24   26
2.33   56      0.025   0.082°    0.574   3.085°    23   38
Steady state** 0.033   0.082°    1.170   2.758     35   34
Deviation ***    15%   0%   11%   18%
        
*   For 1 - 4 hours the means of the concentrations of the
    actual and last sample were used
**  Means were calculated from data marked with "°" 
    considered to represent steady state conditions.
BCFs were calculated from these steady state concentrations.
*** Deviation was calculated as half of the range of those 
    data which were regarded as representing the steady
    state.

Validity criteria fulfilled:
yes
Conclusions:
Depuration
At the end of the study at the sampling points after 72 and 96 h and at both treatment levels the concentrations of 2-Ethylhexyl methacrylate in tissue were below the LOQ, corresponding to more than 99 % depuration at the high test concentration.

Water concentrations and uptake
The 2-Ethylhexyl methacrylate concentrations in the water during the uptake phase (with fish) were considerably lower than those achieved during the system equilibration phase (without fish).
In both treatments the concentrations during the first hours decreased considerably, most probably due to rapid uptake by the high number of initially present fish and adsorption processes. For the period considered for steady state conditions, the 2-Ethylhexyl methacrylate concentrations were within 20 % of the mean measured concentration. Due to the very fast uptake and elimination processes, the concentrations can be regarded as sufficiently constant for determining a reliable BCF. At the low treatment level concentrations in fish decreased, which might indicate an increase of metabolic transformation, being less efficient at the higher treatment level.

Uptake rate constant and BCF
The fitted uptake rate constant k1 was also very similar at both concentrations. The deviation from the mean was approximately 5 % and the standard errors were < 20 % of the mean

Summary of kinetic study results +/- standard error

Deviation
Low treatment High treatment from mean
k1 [1/h] 19.2 +/- 1.1 17.4 +/- 1.6 4.9 %
k2 [1/h] 0.51 +/- 0.09 0.47 +/- 0.03 4.1 %
BCFk 37.5 37.4 0.1 %

As concluding result of the study, a BCF of 37 has been found for 2-Ethylhexyl methacrylate, calculated as mean BCFk derived from the two test concentrations.

This value is near the range of 34 - 35 calculated for the BCFss.
Executive summary:

In a valid guideline study according OECD 305 a BCF of 37 has been found for 2-Ethylhexyl methacrylate, calculated as mean BCFk derived from the two test concentrations. This value is near the range of 34 - 35 calculated for the BCFss.

Description of key information

No study exists for the bioaccumulation of Dodecyl methacrylate.

Read across to structural analogue substance 2-ethylhexyl methacrylate (EHMA):

An experimental study on the structural analogue 2-ethyl hexyl methacrylate (log Kow > 4) (Schäfers, 2006) of bioconcentration

of the substance, according to OECD guideline 305 demonstrates that a rapid metabolisation occurs, this depuration prevents any significant accumulation. The measured BCF is 37.

Key value for chemical safety assessment

BCF (aquatic species):
37 dimensionless

Additional information

No study exists for the bioaccumulation of Dodecyl methacrylate.

Read across to structural analogue substance 2-ethylhexyl methacrylate (EHMA):

The octanol/water partition coefficient of the substances (please see section 4.7), log Kow > 4, predicts a potential for bioaccumulation.

Simple models linking BCF to logKow would give BCF in the range of about > 1000 depending on the model and the logKow used .

On the contrary, an experimental study (Schäfers, 2006) has been carried out on the structural analogue 2-ethylhexyl methacrylate (log Kow > 4) to assess the fish bioconcentration of the substance, according to OECD guideline 305.

This study was conducted with nominal concentrations of 0.06 and 0.3 mg EHMA/l under flow through conditions. An untreated control was run in parallel. Based on a range-finding experiment which indicated rapid metabolism, the uptake phase was scheduled for 56 hours, the depuration phase for 30 hours. Samples of fish (LOQ 0.05 mg/kg) and water (LOQ 0.001 mg/L) were taken at the same time and analysed for 2-Ethylhexyl methacrylate by GC-MS/MS in order to derive the uptake and depuration constants as well as the bioconcentration factor at equilibrium between uptake and elimination. No mortality was observed in the study. During the uptake phase, all concentrations in water and fish were above the LOQ. In the low treatment group, water concentrations rapidly decreased to 50% of nominal during the first 2 h of exposure.

They remained sufficiently constant thereafter. In the high treatment group, water concentrations rapidly decreased to 30% of nominal during the first 4 h of exposure. They remained sufficiently constant thereafter. For steady state calculations the mean of the concentrations in the water of the last five respectively four samplings were used (low 4 – 56 h, high 8 -56 h).

Concentrations in fish increased fast within the first hours and declined later when concentrations in water remained constant. For a worst case estimation, the last two measurements of the concentrations in fish in the low treatment group were not considered further. The steady state BCF was calculated to be 35 and 34 for the low and the high test concentration, respectively. In the depuration phase concentrations in the fish were below LOQ after 16 h. The depuration rates were calculated to be 0.51/h and 0.47/h for the low and the high treatment, respectively.

The depuration half-life is approximately 1.5 h, 95 % is depurated within approximately 6 h. The equilibrium between uptake and depuration (steady state) is already reached after 2 h of exposure. After 12 h, the concentration factor decreased, particularly in the low treatment group and thus was not used for fitting the uptake rate. Uptake rates were calculated to be 19.2/h and 17.4/h for the low and the high test concentration.

From the kinetic rates the BCF of ethylhexyl methacrylate was calculated to be 37 for both test concentrations.

 

Bioavailability of 2-ethylhexyl methacrylate is expected to be higher than dodecyl methacrylate as the molecular weight is little lower (EHMA, C12H22O2: 198.31 g/mol; Dodecyl methacrylate , C16H30O2: 254.42 g/mol). EHMA and Dodecyl methacrylate are both alkyl methacrylates and the same way of rapid metabolism is expected. An exact BCF cannot be estimated but it will also be in the same order of magnitude like EHMA and definitively lower than the criteria for classification and labelling of BCF ≥ 500.