reaction product of diphenylmethanediisocyanate, toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate), octylamine, oleylamine and 4-ethoxyaniline (molar ratio 3.88:1:6.38:0.47:2.91)

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-07 to 2011-10-21

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

adapted 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

Draft Guideline 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Radiolabeled test item:
- Chemical name: Tolyl bis urea compound, [ring-14C], 1,1'-(2,4-toluene)bis(3-(4-ethoxy)phenylurea), [4-ethoxyphenyl-ring-U-14C]
- Lot.: TJBIOS-NB32-47
- Molecular formula: C25H28N4O4
- Molecular weight: 448.5
- Radiochemical purity (%): 99.3
- Chemical purity (%): 98.7
- Specific radioactivity (mCi/mmol): 13
- Storage conditions: Freezer

Non-radiolabeled test item:
- Chemical name: Tolyl bis urea compound, N,N”-(Methyl-1,3-phenylene)bis[N’-p-ethoxyphenyl-urea]
- Lot.: TJBIOS-NB32-46
- Molecular formula: C25H28N4O4
- Molecular weight: 448.5
- Purity (%): 99.1
- Storage conditions: Freezer
- Expiration date: 14 March 2012

Remark: Because of the low solubility of the test item (Urea (2,4-Toluylene diisocyanate + p-Phenetidine)) in solvents (e.g. water, methanol, n-octanol, aceton, toluol and n-heptan) and the low sensitivity of the analytical method available the experimental determination of the solubility of the test item was technically not feasible.

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms: Fish were sampled on test days 5 and 10 after start of the exposure, and on days 1, and 3 after start of the depuration phase (days 11 and 13 after start of exposure). On each sampling date, five fish were sampled from each test group.
- Details on sampling and analysis of test organisms: After sampling, the fish were killed by neck cut. After they were blotted dry the wet weight and length was recorded. On sampling days 10, 11 and 13, the fish were processed immediately after sampling. After measurements, each fish was dissected into viscera and edible parts. The viscera contained the gastrointestinal parts of the fish. These parts were analysed separately for TRR by combustion. The weight of the fish was recorded, but not of the separate parts after dissection.
A total of four fish were sampled on the day of test start from the the culture to determine their dry mass. After the fish were killed using a Tricaine solution, the wet weight was determined. Thereafter, the fish were dried at 60°C to dryness. After cooling in an exsiccator the dry weight was determined.
The lipid content was determined in fish samples collected at beginning of the test (day 0) and on days 10 and 13, representing the end of the exposure phase and the end of the depuration phase, respectively. On day 0, three fish of the culture were analyzed for their lipid content. On day 10 and 13 three fish of each treatment group were collected and pooled per test group before lipid content determination. Fish were kept deep frozen until lipid determination. The method for fat content determination in fish was based on the method of Bligh and Dyer (1959).

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Two stock solutions with radiolabeled test item were prepared. The first stock solution was prepared by dissolving a small amount of radiolabeled test item in 5 mL of acetone. The solution was not clear but homogenous. The concentration of test item was determined to be 107.7 µg test item/mL using triplicate 25 µL aliquots for LSC. Due to an error in food calculation and preparation another stock solution had to be prepared. The second stock solution was prepared by dissolving a small amount of radiolabeled test item in 10 mL of acetone. The solution was not clear but homogenous. The concentration of test item was determined to be 175.4 µg test item/mL using triplicate 10 µL aliquots for LSC. The final stock solution with radiolabeled test item was prepared by mixing the freshly prepared solution with the remaining solution of the first prepared radiolabeled stock solution. The solution was analyzed for its concentration using triplicate 20 µL aliquots for LSC to be 159.7 µg test item/mL. A stock solution with non-radiolabeled test item was prepared by weighing 23.9 mg of the non-radiolabeled test item into a 20 mL volumetric flasks and dissolving it in acetone. The concentration of the non-radiolabeled stock solution was therefore 1.195 mg test item/mL. The test item was not clear but the solution was sonicated for homogenous distribution of the test item. The food of the treatment group was prepared using a total of 26.25 g of grinded food. The stock solutions with radiolabeled test item and with non-radiolabeled test item were added to the food powder, i.e. a total of 2.4 mg of radiolabeled and 23.9 mg of non-radiolabeled test item were applied. The concentration of test item in food was therefore 1.00 mg/g. The volume of total 35 mL of acetone was allowed to evaporate off at room temperature. The fish food was stirred from time to time during the evaporation to ensure homogenous distribution. The applied food/solvent ratio was 1 g/ 1.33 mL. The solvent was allowed to evaporate for total dryness of the food, therefore, no solvent was left in the food.
- Chemical name of vehicle: Acetone
- Solvent control: The food for the solvent control was prepared using a total of 25.18 g grinded food and 25.5 mL of acetone. The applied food/solvent ratio was approximately 1 g/ 1 mL. The solvent was allowed to evaporate for total dryness of the food, therefore, no solvent was left in the food.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Osage Catfisheries, Inc. 1170 Nichols Road, Osage Beach, MO 65065, USA
- Age at study initiation (mean and range, SD):
- Length at study initiation (lenght definition, mean, range and SD): 5.0 ± 2.0 cm
- Weight at study initiation: two weight classes: 1.14 - 1.73 g and 1.73 - 1.87 g
- Weight at termination (mean and range, SD):
- Health status: healthy
- Food type: commercially available fish food diet Hikari Staple was used
- Amount: 2.5 % of the wet body weight of the fish
- Frequency: once a day

ACCLIMATION
- Type and amount of food: commercially available fish food diet Hikari Staple was used
- Acclimation conditions: same as in the main test
- Feeding frequency: daily
- Health during acclimation (any mortality observed): mortality was less than 1 %

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

10 d

Total depuration duration:

6 d

Hardness:

0 to 44 mg/L

Test temperature:

20.0 to 23.1 °C

pH:

7.25 to 7.82

Dissolved oxygen:

above 60 % of saturation

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 L stainless steel test vessles (62 cm x 50 cm x 20.5 cm)
- Material, size, headspace, fill volume:
- Aeration: yes, using glass pipettes connected to a pump
- Renewal rate of test solution (flow rate): the flow rate was set to 278 mL/minute
- No. of organisms per vessel: 61 fish per vessel
- No. of vessels per concentration (replicates): one replicate
- No. of vessels per control / vehicle control (replicates): one replicate
- Biomass loading rate: 0.22 g/L/day

TEST MEDIUM / WATER PARAMETERS
- according to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark (except the 30 min transition period)
- Light intensity: 200 to 248 Lux

PRELIMINARY STUDY
- Test concentrations: To evaluate the toxicity of the test item to fish, a preliminary acute toxicity study over 96 hours was performed with 10 mg test item/g diet, which was 10 fold the intended concentration of 1 mg test item/g diet for the definitive dietary bioaccumulation study.
- Results used to determine the conditions for the definitive study: No mortality was observed within the 96 hours acute toxicity study. The concentration of 10 mg test item/g food showed no toxic effects on fish. Based on these results, the definitive study was performed with a concentration of 1 mg test item/g food.

Nominal and measured concentrations:

1 mg test item/ g food (nominal concentration)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

No absorption of the test item in fish was observed. No BMF or degradation values could be calculated.

Lipid content:

29.8 %

Time point:

start of exposure

Lipid content:

37.6 %

Time point:

end of exposure

Lipid content:

22.3 %

Time point:

other: Day 3 of Depuration

Remarks on result:

other: No BMF or degradation values could be calculated.

Elimination:

no

Details on kinetic parameters:

The chemical assimilation efficiency α, the uptake or depuration rate constant could not be calculated due to the fact that no uptake of the test item in fish was observed and therefore also no depuration. The calculation of the dietary biomagnification factor as well as the lipid normalized biomagnification factor was not possible, either.

Details on results:

- Mortality of test organisms: In the treatment group the mortality was at 4.9% with 3 dead fish.
- Behavioural abnormalities: The fish did not display any signs of effects resulting from the test item throughout the study period.
- Observations on body length and weight: The fish lost weight over the course of the study. In the solvent control, the fish measured at test end showed only 90.7% of the initial weight at test start. In the treatment with test item, the weight at test end represented 94.4% of the weight at test start. The difference between the solvent control and the treatment group was within 4% of each other, indicating that no influence of the test item was observed on the growth of the fish. Due to the fact that the fish lost weight over the course of the study, no growth curve can be given.
- Results with vehicle control: Mortality in the solvent control was 9.8%, with 6 dead fish of a total of 61 fish.

Validity criteria fulfilled:

yes

Conclusions:

No absorption or biomagnification of test item in fish was observed. Therefore the test item has no bioaccumulation potential.

Executive summary:

Pimephales promelas were exposed for 10 days to diet treated with test item (treatment group) or solvent (solvent control), under flow-through conditions. The depuration phase with untreated food was performed for 3 days. The flow rate per test vessel was approximately 278 mL/minute (approximately 400 L/day) corresponding to 8 volume exchanges of the test vessel per day. One replicate was set up per test group, i.e. one replicate as solvent control and one replicate as treatment group. The test vessels were stainless steel vessels with a volume of 50 L. Fish were sampled on days 5 and 10 after start of the exposure. During the depuration phase, fish samples were collected on days 1 and 3 after start of the depuration phase, i.e., on test days 11 and 13 after start of the exposure. On each sampling interval, five fish were sampled from each test group. Fish were used to recalculate the amount of food required to maintain a feeding rate of 2.5% of their wet body weight. Total radioactive residues were determined in the fish of sampling intervals day 10, 11 and 13. Fish were observed daily for mortality and adverse effects. Operation of the dilutor was checked daily and the temperature was measured continuously in the test solution of the solvent control. Regular measurements of the dilution water were conducted, including dissolved oxygen concentration, pH, alkalinity and hardness. The mortality in both test groups was below the triggered 10% limit. Analyses of the food confirmed that the test item was correctly dosed and the food was stable over the course of the study. All validity criteria were fulfilled. No absorption of the test item in fish was observed. No BMF or degradation values could be calculated.

Description of key information

The bioaccumulation potential of the test substance was assessed in a flow–through fish test using the model substance Tolyl bis urea compound, as agreed between The German Federal Environmental Agency (Umweltbundesamt), Klüber Lubrication and SCC GmbH. This decision was confirmed by the Federal Institute for Occupational Safety and Health (BAuA) on the 26th of April in 2008 (letter attached). Fish were exposed for 10 days to diet treated with 1 g/kg test item. The 10 days uptake phase was followed by a 6 days depuration phase. The test demonstrates that the test item has no bioaccumulation potential.   

Additional information

Pimephales promelas were exposed for 10 days to diet treated with test item (treatment group) or solvent (solvent control), under flow-through conditions. The depuration phase with untreated food was performed for 3 days. The flow rate per test vessel was approximately 278 mL/minute (approximately 400 L/day) corresponding to 8 volume exchanges of the test vessel per day. One replicate was set up per test group, i.e. one replicate as solvent control and one replicate as treatment group. The test vessels were stainless steel vessels with a volume of 50 L. Fish were sampled on days 5 and 10 after start of the exposure. During the depuration phase, fish samples were collected on days 1 and 3 after start of the depuration phase, i.e., on test days 11 and 13 after start of the exposure. On each sampling interval, five fish were sampled from each test group. Fish were used to recalculate the amount of food required to maintain a feeding rate of 2.5% of their wet body weight. Total radioactive residues were determined in the fish of sampling intervals day 10, 11 and 13. Fish were observed daily for mortality and adverse effects. Operation of the dilutor was checked daily and the temperature was measured continuously in the test solution of the solvent control. Regular measurements of the dilution water were conducted, including dissolved oxygen concentration, pH, alkalinity and hardness. The mortality in both test groups was below the triggered 10% limit. Analyses of the food confirmed that the test item was correctly dosed and the food was stable over the course of the study. All validity criteria were fulfilled. No absorption of the test item in fish was observed. No BMF or degradation values could be calculated.