Bis(2,6-diisopropylphenyl)carbodiimide

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-11 - 2021-04-09 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test

Version / remarks:

OECD (2012). Bioaccumulation in fish: aqueous and dietary exposure. OECD Guidelines for testing of chemicals, Section 3, OECD Publishing, Paris, test No. 305

Deviations:

yes

Remarks:

Two deviations occurred during the test. However, these deviations had no effect on the quality and validity of the test. For further information please refer to section "Any other information on materials and methods incl. tables".

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms: Fish of both groups, control and treatment, were sampled twice during the uptake phase (days 12 and 14) and six times during depuration (1, 3, 7, 10, 14, 21 days).
The specific time points refer to the start of each phase, which was initiated by feeding the respective diet. The uptake phase started with feeding spiked feed, the depuration with feeding unspiked feed.
- Sampling intervals/frequency for test medium samples: Feed samples for chemical and lipid analysis were taken on day 0 and 14 of the uptake phase.
- Sample storage conditions before analysis: All samples were stored at <-18°C until further processing.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Samplings were conducted before the daily feeding routine of the remaining test population. For all samplings, fish were anesthetized in MS 222, rinsed, blotted dry and shock-frozen in liquid nitrogen after recording animal parameters (length, weight).

Fish samples for chemical analysis: At each sampling event, five specimen of both populations were sacrificed for matrix collection (complete animal) and subsequent analytical assessment. Each fish was analyzed individually for the test item to generate five replicates per sampling date. One control fish was sampled at test start to monitor a background contamination in the animals used for experimentation. Further control fish sampled at the end of uptake and end of depuration were analyzed first with only one replicate of the sampled specimen to monitor contaminations during experimentation.
For analysis of concentration in fish the whole fish were cut, homogenized and extracted with ACN. The extractable radioactive residues (ERR) were assessed by liquid scintillation counting (LSC). The post-extraction solid was treated with tissue solubiliser and the amount of the non-extractable radioactive residues (NER) where quantified by means of LSC. Total radioactive residues (TRR) were calculated as the sum of ERR and NER. Further, the amount of CDI was assessed by parent-specific analysis with radio-thin layer chromatography (TLC) of the extracted radioactivity. Also for fish the specific tissue concentration was calculated based on the recorded weight data.

Fish samples for lipid analysis: Three reference fish from the stock population were sampled at the beginning of the experiment to determine a reference lipid content. Thereafter, three specimen of each test population were sacrificed for monitoring lipid contents at the end of the uptake and at the end of the depuration phase. The lipid fractions were determined gravimetrically after extraction according to a method from Smedes (SigmaPlot® 13.0, Systat Software Inc., 2014).

Feed samples for chemical analysis: Triplicate samples of test and control diet were taken for chemical analysis.The concentration of the test substance in feed was assessed prior to the first feeding and at the end of the uptake period, respectively. Here, small portions of approximately 1 g were homogenized and extracted with ACN using an ULTRA-TURRAX®. Subsequently, the samples were analysed by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) after appropriate dilution. The specific test concentration on feed was then calculated based on the recorded weight data.

Feed samples for lipid analysis: Triplicate samples of test and control diet were nalysed for the lipid content. All lipid samples were analysed for total lipid content according to a method by Smedes (SigmaPlot® 13.0, Systat Software Inc., 2014).

Vehicle:

yes

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

- Details on fish food (source, fat content as supplied, etc): Commercial fish feed with a pellet size of 2 mm was used for experimentation.
- Details of spiking (e.g. i) liquid test material (neat); ii) with a vehicle (corn or fish oil); or iii) using an organic solvent: The test item was applied by solvent spray application with a targeted nominal test concentration of 10 mg/kg. Prior to the application of the test item, the feed was coated with sulfuric acid to methylate all superficial carboxylic acid functionalities. Therefore, 200 g of the feed pellets were placed in a 2 L pear-shaped flask connected to a rotary evaporator and equipped with a stainless steel capillary to apply the sulfuric acid solution via a solvent-inlet tube to the pellets under vacuum. 20 mL of the freshly prepared coating solution (= 1 % sulfuric acid in MeOH) was spread among the feed pellets, which were being constantly mixed by rotation to ensure a homogenous distribution of the solution on the pellets. During this coating process, a low pressure of approximately 600 mbar at the rotary evaporator was set. The coating solution containing vessel was rinsed with 1 mL MeOH and this additional volume was also applied to the feed for rinsing the capillary.
For application of the test item, 100 g of the coated fish feed was placed again in a 2 L pear-shaped flask and was connected to the rotary evaporator equipped with the stainless steel capillary. As a typical loss of about 15 % can occur during the procedure of the spray application, 15 % of the test item was added to the pursued 10 mg/kg. Thus 1.15 mg of CDI were applied to the preserved 100 g sulfuric acid coated test feed. An application solution was prepared by mixing 845 µL of the radioactive stock solution (1.36 mg/mL, for preparation see A.3.4) with 2.2 mL MeOH. This application solution was then spread among the feed particles through spray application, while the feed being constantly mixed by rotation to ensure the homogenous distribution of test item onto the pellets. During spiking, a low pressure of approximately 600 mbar was set to run the application. The test item containing vessel was rinsed with 1 mL MeOH, thrice. This additional volume was also applied to the feed for rinsing the vessel and the capillary. After that, the vacuum of the rotary evaporator was set to 400 mbar for another 15 min to remove the solvent. The control feed (for uptake) was prepared the exact same way but without the test item, here 91 g test feed were applied with 6 mL pure MeOH. Afterwards, the spiked pellets were stored at ≤ -18 °C.
As the fish weight and thus the size of the feed portions for the first week of uptake was determined at test start, the feed batch was thawed and specific portions for the first week were weighed to avoid repeated freeze-thawing cycles. After the first week of experimentation, the daily feed ration was adjusted to the increased body weight and thus, the feed portions for the second week were weighed in after thawing of the feed batch. For smaller feed portions (e.g. from day 12 on after sampling and other occasions (e.g. escape from animals)), the small weight adjustments of the portions were directly performed before feeding.
The homogeneity and content of test and control feed was analysed one day after preparation.
- Chemical name of vehicle (organic solvent), if used: MeOH
- Method for removal of solvent, if used: The vacuum of the rotary evaporator was set to 400 mbar for approximately 15 min to remove the solvent. Subsequently, the acid coated pellets were incubated at 55 °C in a drying cabinet overnight in order to remove potential solvent residues.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss, Teleostei, Salmonidae)
- Source: Fischzucht Störk, Wagenhausen 8, 88348 Bad Saulgau, Germany
- Age at study initiation (mean and range, SD): juvenile specimen
- Length at study initiation (length definition, mean, range and SD): 7.45 ± 0.42 cm (determined by measuring of 25 individuals of control and treatment vessel)
- Weight at study initiation (mean and range, SD): 4.08 ± 0.66 g (determined by measuring of 25 individuals of control and treatment vessel)
- Weight at termination (mean and range, SD): 9.86 ± 1.29 g
- Method of breeding: The fish were held in water of the same quality as used in the test (purified drinking water) until the start of exposure. Test fish were held for at least 14 days prior to the test under equivalent water quality and illumination conditions to those used in the test. Fish were fed ad libitum throughout the holding period with trout feed once daily.
- Lipid content at test initiation (mean and range, SD): 6.13 ± 0.366 %
- Health status: Only healthy fish, free from observable diseases and abnormalities were used in the study. Mortality of the batch was less than 5 % in the week preceding the start of the study.
- Feeding during test
- Food type: commercially available trout feed pellets
- Amount: 2.0 % of the body weight per day
- Frequency: daily

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Test fish were held under equivalent water quality and illumination conditions to those used in the test.
- Type and amount of food: Fish were fed ad libitum throughout the holding period with trout feed.
- Feeding frequency: once daily
- Health during acclimation (any mortality observed): Only healthy fish, free from observable diseases and abnormalities were used in the study. Mortality of the batch was less than 5 % in the week preceding the start of the study.

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

21 d

Hardness:

total hardness: 6.01 °d (mmol/L)
Ca-hardness: 5.0 °d (mmol/L)
Mg-hardness: 1.0 °d (mmol/L)

Test temperature:

13.8 - 14.6 °C

pH:

7.21 and 7.72

Dissolved oxygen:

content: 7.50 - 9.21 mg/L
Air saturation value: 81.5 - 103 %

TOC:

control: mean 0.690 mg/L ± 0.912 (NPOC level in water at the beginning of the study)
treatment: mean 0.674 ± 0.929 (NPOC level in water at the beginning of the study)

Salinity:

not applicable

Conductivity:

264 µS/cm

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 L glass aquaria
- Material, size, headspace, fill volume: 75 L fill volume
- Aeration: The test vessels were aerated via a glass capillary connected to an air pump.
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous flow using a metering pump system
- Renewal rate of test solution (frequency/flow rate): a flow rate of at least 15.6 L/h water was maintained
- No. of organisms per vessel: 49
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: A maximum fish-to-water loading rate of 0.1 to 1.0 g fish (wet weight) per liter of water per day was not exceeded throughout the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-chlorinated local tap water was used in accordance with the OECD guidelines. For details please refer to section "Any other information on materials and methods incl. tables".
- Holding medium different from test medium: no
- Intervals of water quality measurement: The following water chemistry data, recorded monthly in the laboratory of the testing facility, were reported: temperature, pH, conductivity [µS/cm], dissolved oxygen content [% ASV or mg O2/L], total residual chlorine [mg/L], content of nitrate [mg/L], nitrite [mg/L], ammonium [mg/L], phosphate [mg/L], magnesium [mmol/L], total hardness [mmol/L], calcium hardness [mmol/L], alkalinity [mmol/L], NPOC content [mg/L], metal content (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) [µg/L].

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: information not available yet
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: 2.0 % of the body weight per day.
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): The fish were fed once at approximately the same time at each day.
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used: control food: 24.5 ± 0.45 %, spiked food: 24.0 ± 0.26 %
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used: control food: 24.3 ± 0.26 %, spiked food: 24.5 ± 0.38 %

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 1 and 10 mg/kg

Nominal and measured concentrations:

nominal: 10 mg/kg feed
measured: 8.02 ± 1.13 mg/kg feed

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: measured: > 6.2, calculated: 8.72 (at 25 °C)

Lipid content:

>= 5.83 - <= 6.54 %

Time point:

start of exposure

Remarks on result:

other: Fish lipid content of the stock

Remarks:

Mean value: 6.13

Lipid content:

>= 7.47 - <= 8.22 %

Time point:

other: End of uptake or depuration start after 14 days

Remarks on result:

other: Fish lipid content of the treatment group

Remarks:

Mean value: 7.88 %

Lipid content:

>= 7.55 - <= 9.31 %

Time point:

other: End of uptake or depuration start after 14 days

Remarks on result:

other: Fish lipid content of the control

Remarks:

Mean value: 8.30 %

Lipid content:

>= 8.52 - <= 10.1 %

Time point:

other: End of depuration after 35 days

Remarks on result:

other: Fish lipid content of the treatment group

Remarks:

Mean value: 9.41 %

Lipid content:

>= 7.25 - <= 9.27 %

Time point:

other: End of depuration after 35 days

Remarks on result:

other: Fish lipid content of the control

Remarks:

Mean value: 7.92 %

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

other: BMFkgl (lipid normalised and growth corrected BMF)

Value:

0.194 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other: Based on measured concentrations of the parent compound

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

BCF

Value:

10 251 dimensionless

Basis:

other: Growth and lipid corrected BMF (BMFkgl)

Calculation basis:

kinetic

Remarks on result:

other: Based on measured concentrations of the parent compound; worst-case value of estimation method 1 according to several calculation references (range: 1020.1 - 10251.0)

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

BCF

Value:

4 917.1 dimensionless

Basis:

other: Growth and lipid corrected BMF (BMFkgl)

Calculation basis:

kinetic

Remarks on result:

other: Based on measured concentrations of the parent compound; estimation method 2 according to Brookes and Crooke (2012)

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

BCF

Value:

3 390.8 dimensionless

Basis:

other: Growth and lipid corrected BMF (BMFkgl)

Calculation basis:

kinetic

Remarks on result:

other: Based on measured concentrations of the parent compound; estimation method 3 according to Inoue et al. (2012)

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

other: BMFkgl (lipid normalised and growth corrected BMF)

Value:

0.479 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Key result

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

BCF

Value:

14 631.8 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption; worst-case value of estimation method 1 according to several calculation references (range: 1456.0 - 14631.8)

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

BCF

Value:

7 595.4 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption; estimation method 2 according to Brookes and Crooke (2012)

Conc. / dose:

8.02 other: mg/kg food

Temp.:

>= 13.8 - <= 14.6 °C

Type:

BCF

Value:

7 166.7 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption; estimation method 3 according to Inoue et al. (2012)

Elimination:

yes

Parameter:

DT50

Remarks:

substance-specific half-life

Depuration time (DT):

8.52 d

Remarks on result:

other: Based on measured concentrations of the parent compound

Elimination:

yes

Parameter:

DT50

Remarks:

growth corrected substance-specific half-life

Depuration time (DT):

13.8 d

Remarks on result:

other: Based on measured concentrations of the parent compound

Key result

Elimination:

yes

Parameter:

DT50

Remarks:

growth corrected substance-specific half lief

Depuration time (DT):

19.8 d

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Elimination:

yes

Parameter:

DT50

Remarks:

substance-specific half-life

Depuration time (DT):

10.5 d

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Rate constant:

growth rate constant (d-1)

Value:

0.031

Rate constant:

overall depuration rate constant (d-1)

Value:

0.081

Remarks on result:

other: Based on measured concentrations of the parent compound

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.05

Remarks on result:

other: Based on measured concentration of the parent compound

Rate constant:

growth-corrected half-life (d)

Value:

13.8

Remarks on result:

other: Based on measured concentrations of the parent compound

Rate constant:

other: assimilation efficiency

Value:

0.159

Remarks on result:

other: Based on measured concentration of the parent compound

Rate constant:

overall depuration rate constant (d-1)

Value:

0.066

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.035

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Rate constant:

growth-corrected half-life (d)

Value:

19.8

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Rate constant:

other: assimilation efficiency

Value:

0.274

Remarks on result:

other: Based on total radioactive residues (TRR) as worst-case assumption

Metabolites:

TLC-analysis of the fish samples showed that no major metabolites were found at the end of the uptake phase (highest tissue concentration). Only on days 10 and 14 of depuration 15.2 % and 6.80 % of TRR remained at the origin of the TLC, respectively. As the amount of radioactivity remaining at the origin on day 21 of depuration was again less than 5 % (2.47 %) it is supposed to not be likely that a major metabolite of CDI was formed in fish matrix.

Results with reference substance (positive control):

not applicable

Details on results:

- Mortality of test organisms: 8.33 % in the control and 0 % in the treatment
- Behavioural abnormalities: Fish of the control group developed a hierarchical behavior during the depuration phase and one fish jumped out of the vessel, two fish were removed dead and another two fish were removed after showing physical injuries. Also, four fish of the treatment group jumped out of the test vessel and were excluded from further experimentation.Thus, the total numbers of fish assessed in the test were corrected from 49 to 48 in the control and from 49 to 45 in the treatment.

Reported statistics:

The kinetic biomagnification parameters were calculated accordingly to the suggested mathematical operations given in the OECT TG 305 for BMF determination using Microsoft Excel 2016® and SigmaPlot® .

Data evaluation

In this report numerical values are frequently rounded to a smaller degree of precision (number of digits) than used in the actual calculation. Minor differences in results or percentages < 100 % obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern.

 

Results

Fish health and behaviour

All fish were in a vivid and healthy condition before and during the uptake phase. Fish of the control group developed a hierarchical behavior during the depuration phase and one fish jumped out of the vessel, two fish were removed dead and another two fish were removed after showing physical injuries. Also, four fish of the treatment group jumped out of the test vessel and were excluded from further experimentation.

Thus, the total numbers of fish assessed in the test were corrected from 49 to 48 in the control and from 49 to 45 in the treatment. For mortality assessment 4 fish in the control and 0 in the treatment were considered, resulting in % mortalities of 8.33 % in the control and 0 % in the treatment.

 

Concentration of CDI in test feed

The test feed yielded a concentration of 8.02 ± 1.13 mg/kg CDI, while the control feed did not contain any trace of test item (values all < LOQ). The homogenous distribution was tested in five replicates before test start. The relative standard deviation (RSD) of 14.1 % indicated an even distribution of CDI on feed and thus approved homogeneity (required by OECD 305: 15 %).

At the end of the uptake phase the test feed was analysed again in triplicates and approved a sufficient stability of CDI on the test feed over time, as indicated by a recovery of 101 % of the initial concentration (required by OECD 305: ± 20 %).

For further information please refer to the attached tables presenting the analytical results in feed samples.

 

Concentration in fish

The concentration of CDI in fish was determined by measurement of radioactive residues in the fish sample extracts (ERR) by LSC and subsequent radio-TLC analysis to quantify the test item based on the area under curve percentages with regard to the injected radioactivity.

In the treatment group a mean tissue concentration of c_14d,CDI = 0.322 ±0.073 mg/kg CDI was determined in fish matrix at day 14, the end of the uptake phase.

A broad sampling schedule was designed for the depuration phase to record the elimination. A starting concentration c_0,d,CDI = 0.213 mg/kg was calculated at the onset of depuration by linear regression of ln-transformed fish concentrations during the depuration period.

TLC-analysis of the fish samples showed that no relevant metabolites were formed (e.g.:Figure20, highest tissue concentration).

The method for the quantification of CDI was assessed as valid. Nevertheless, as radioactive material was used, it was possible to gain a broader insight into the bioaccumulating potential of test item related residues. Thus, two additional perspectives were evaluated. Notably, those evaluations constitute worst case considerations and could only be made due to the usage of radiolabeled material.

The first consideration is based on the complete extractable fraction (no parent specific analysis) and the second, as an absolute worst case scenario, is based on total radioactive residues, resulting from the sum of the ERR and NER.

Here, mean tissue concentrations of c_14d,ERR = 0.378 ±0.079 mg/kg and c_14d,TRR = 0.378 mg/kg were determined in fish matrix, for ERR and TRR, respectively at the end of the uptake phase.

The tissue concentrations at the onset of depuration were calculated by linear regression of ln-transformed fish concentrations during the depuration period with c_0,d,ERR = 0.258 mg/kg and c_0,d,TRR = 0.401 mg/kg.

The elimination of the test item to < 95 % was not achieved during the three weeks of depuration. Based on parent specific CDI tissue concentration, an elimination of 85.4 % to 14.6 % of CDI was achieved. In terms of total radioactive residues elimination up to 21.1 % was achieved.

The stock population, as well as the analyzed control fish, were free of pre- or cross-contaminations. All respective samples resulted in values <LOQ.

For further information please refer to the attached tables presenting the analytical results in fish samples and the attached figure presenting the absolute tissue concentrations in fish during the study.

 

Lipid correction

The lipid content of fish from the stock population was about 6.13 %. Over the experimental phase of the study, a mean lipid content of 7.45 % was determined in control animals and 7.81 % in the exposed specimen (see Annex A.2.3). The slight increase compared to the stock population was due to differing of feeding rates, with which fish were fed in culture (approximately 1.5 % feed of body weight per day) or during experimentation (2 % feed of body weight per day). The fish diet contained 24.3 % lipids in control diet as well as in the spiked diet (see Annex A.2.3). The used feed is therefore very similar to the typical feed recommendation of 15 - 20 % lipid in diets. For BMF-normalization, a lipid correction factor of L_c=0.326 was calculated.

A figure presenting the ln-linear fit of the fish matrix concentrations during depuration is included as attachment.

 

Growth correction

Fish were measured and weighted at the beginning of the experiment as well as at respective sampling time points to monitor growth to monitor associated growth-dilution effects during experimentation. This physiological parameter is an important measure to detect potential adverse effects that may occur with differing diets. Growth rate constants were determined separately for the uptake and depuration phases, for the control and treatment group, using the ln-transformed weights of the fish. A subsequent Parallel Line Analysis (PLA, as suggested by the TG) resulted in a statistical difference between the uptake and depuration phase among control animals (P = 0.015). For the test item-treated group, no difference between uptake and depuration was detected (P = 0.319). Thus, growth data from the depuration phase were compared between control and treatment, where no statistical difference was detected (P = 0.549). The data were pooled to derive the overall fish growth rate constant kg. The value was determined with kg= 0.0313 per day(R2 = 0.576) and was then used for growth correction of the depuration rate constants as recommended by OECD TG 305 (subtraction from k2).

The growth data indicate that no adverse or toxic effects were caused by the diet with the test item.

A figure presenting the ln-transformed weight data of control and treated fish during depuration is included as attachment.

 

The tables presenting the biological raw data are included as attachment for full information also.

 

Validity of the test

Concerning the validity criteria given in the OECD guideline 305:

1. Water temperature variations were in the range of the suggested 15 ± 2 ºC as suggested by the OECD 305.


2. The concentration of dissolved oxygen did not fall below 60 % of the air saturation in water.


3. The stability of the test item-spiked feed was confirmed to be within a range of ± 20 % when comparing the concentration of five replicate samples of test feed at the start of the study with triplicates after completion of the uptake phase.


4. A high degree of homogeneity of substance on test feed was demonstrated before test start based on five replicates which did not vary more than ± 15 % from their mean concentration.


5. Control fish before study, at end of uptake and end of depuration phase did not show any contamination and thus, were free of test item.


6. No adverse effects or test-related mortalities or diseases were recorded during the study.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD 305-III (Dietary exposure Bioaccumulation Fish Test) with the registered substance. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the bioaccumulative potential of the registered substance towards fish.
Due to the test substance characteristics, the registered substance was administered to a test population of fish via feed at a concentration of 8.02 ± 1.13 mg/kg test substance in accordance to the conditions of the OECD TG 305 for BMF studies.
The test duration was 14 days for the uptake and another 21 days for the depuration phase. No adverse effects were observed during the time course of the study. The test item concentration at the sampling points was used to assess the elimination kinetics of CDI in fish matrix. Due to the usage of radiolabelled material additional worst case considerations could be made based on the complete extractable fraction (ERR, no parent specific analysis) and, as an absolute worst case scenario, based on total radioactive residues (TRR), resulting from the sum of the ERR and NER. Accordingly, the BMFKGL was determined to be 0.194 for the parent compound, 0.251 for ERR and 0.479 for TRR.
By the use of the spreadsheet provided in conjunction with the Guidance Document to OECD TG 305, a BCF was estimated for the registered substance as well as for the TRR as worst-case assumption. Accordingly, a BCF of 10257.0 was determined for the parent compound (range 1020.7 - 10257.0) and a worst-case BCF of 14631.8 was determined for TRR (range 1456.0 - 14631.8). Hence, it can finally be concluded that the registered substance has a very bioaccumulative potential in the aquatic environment.

Executive summary:

A GLP study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the biomagnification factor of the registered substance in the fish species Oncorhynchus mykiss.

 

The registered substance was administered to a test population of fish via feed in accordance to the conditions of the OECD TG 305 for BMF studies.

The suitability of aqueous exposure of the test item to be applied in a BCF study according to OECD TG 305 was tested and evaluated in the test facility of Fraunhofer IME (see statement ‘Suitability of an aqueous exposure for a BCF study with Bis(2,6-diisopropylphenyl)carbodiimide (non-GLP pre-test)’ dated on March 27, 2019 as attached background material). The results on the low stability of the test item in the aqueous phase provided clear indications that an aqueous exposure is not practicable.

According to OECD TG 305, a dietary exposure test can alternatively be used for substances with high hydrophobicity (test item log K_OW= 6.2) and low water solubilities (test item solubility < 0.53 µg/L). Thus, the dietary approach, generating a biomagnification factor (BMF) was further investigated to evaluate of the bioaccumulation potential of carbodiimide.

 

A test concentration of 8.02 ± 1.13 mg/kg CDI was achieved by applying solvent-mediated spiking on commercially available feed pellets. The feed pellets were pretreated by methylation of external carboxylic acid functionalities.

 

In addition, a control population of fish was fed with similar prepared feed (solvent-spiked) without the test substance to monitor potential mortalities or other adverse effects over time. Both feed types were administered with a rate of 2 % mean body weight per day.

 

The test duration was 14 days for the uptake and another 21 days for the depuration phase. No adverse effects were observed during the time course of the study.

 

Repeated samplings were done for both fish populations and five individuals were picked at each time point for chemical analyses. During the uptake phase, fish were sampled twice, at days 12 and 14 and the highest mean CDI concentration in fish was 0.322 ± 0.073 mg/kg at day 14.

 

During depuration the fish were sampled six times, at day 1, 3, 7, 10, 14 and 21 day of depuration. The test item concentration at the sampling points was used to assess the elimination kinetics of CDI in fish matrix. Based on that, a depuration rate constant of k₂ = 0.0814 was calculated.

 

With that a substance specific half-life (t_1/2) of 8.52 days could be determined.

 

The test item concentration on feed, k₂and a test item concentration of c_0,d = 0.213 mg/kg in fish at the beginning of the depuration phase was used to determine an assimilation efficiency (α) of 0.159.

 

Based on the assimilation efficiency and the feed ingestion rate of 0.02 a kinetic BMF_Kof 0.0391, a growth-corrected BMF_Kgof 0.0634 and a growth- and lipid-corrected BMF_KgLof 0.194 was specified for the registered substance.

As radioactive material was used in this study, it was possible to gain a broader insight into the bioaccumulating potential of test item related residues. Thus, two additional perspectives were evaluated. Notably, those evaluations constitute worst case considerations and could only be made due to the usage of radiolabeled material.The first consideration is based on the complete extractable fraction (ERR, no parent specific analysis) and the second, as an absolute worst case scenario, is based on total radioactive residues (TRR), resulting from the sum of the ERR and NER.

For ERR and TRR mean tissue concentrations of c_14d,ERR = 0.378 ±0.079 mg/kg and c_14d,TRR = 0.378 mg/kg were determined in fish matrix, respectively at the end of the uptake phase. Based on the analytical measurements during the depuration phase and the assessment of the elimination kinetics, the depuration rate constant (k₂) was determined to be 0.0774 for ERR and 0.0663 for TRR with substance specific half-lifes (t_1/2) of 8.96 and 10.5 days, respectively.

Accordingly, the assimilation efficiency was determined to be 0.159 for ERR and 0.274 for TRR, resulting in final growth - and lipid-corrected BMF_KgLof 0.251 for ERR and 0.479 for TRR.

By the use of the spreadsheet provided in conjunction with the Guidance Document to OECD TG 305, a BCF was estimated for the registered substance as well as for the TRR as worst-case assumption. Taking into account that the registered substance is not molecularly large containing two not halogenated aromatic rings only, the logKow was experimentally determined to be 6.2 and the assimilation efficiency within the experimental bioaccumulation study was determined to be 0.159 for the parent compound, the test substance falls within the applicability domain of the first and second BCF estimation method as outlined in the respective guidance document to OECD TG 305 (Table 4 -14). The result obtained by estimation method 3 is considered to be not reliable accordingly, as the test substance is not within its indicative applicability domains.

 

Hence, the results obtained by estimation method 1 and 2 are considered most meaningful with a worst-case BCF of 10251.0 for the parent compound (range 1020.1 - 10251.0) and 14631.8 for TRR (range 1456.0 - 14631.8). Thus, it can finally be concluded that the registered substance has a very bioaccumulative potential in the aquatic environment.

Reference 2

Endpoint:

bioaccumulation: aquatic / sediment

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

16 SEP 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR toolbox v4.4.1

2. MODEL (incl. version number)
BCFBAF v3.01

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)c1cccc(C(C)C)c1N=C=Nc1c(cccc1C(C)C)C(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please refer to the QMRF attached under 'Attached justification'.

5. APPLICABILITY DOMAIN
Please refer to the QPRF attached under 'Attached justification'.

Guideline:

other: REACH guidance on QSAR R.6, May 2008

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR toolbox v4.4.1, EPI Suite v4.11
- Model(s) used: BCFBAF v3.01
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

Specific details on test material used for the study:

CC(C)c1cccc(C(C)C)c1N=C=Nc1c(cccc1C(C)C)C(C)C

Type:

BCF

Value:

1 912 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Regression-based estimate (Traditional method)

Type:

BCF

Value:

1 209 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas method, based on mechanistic first principles

Conclusions:

The bioconcentration factor was predicted using the US-EPA software BCFBAF v3.01. Using the regression-based estimate (traditional method) a BCF of 1912 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 1209 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 59.9 days, whereby the rate constant (kM) for 10 g fish is designated as 0.01158/day. The prediction falls in the applicability domain of the model.

Executive summary:

The prediction for the bioconcentration factor (BCF) of the substance bis-(2,6-diisopropylphenyl)carbodiimide was determined by the computer program BCFBAF v3.01 (EPIWIN software) by US-EPA (2017). Furthermore the whole body primary biotransformation rate estimation for fish was calculated with the notation that the bio half-life normalized to 10 g fish at 15 °C. It is possible to predict the apparent metabolism half-life in fish for three different trophic levels (lower, mid and upper). Using the regression-based estimate (traditional method) a BCF of 1912 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 1209 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 59.9 days, whereby the rate constant (kM) for 10 g fish is designated as 0.01158/day. This is taken into account to predict the apparent metabolism half-life in fish for the substance. For the lower trophic level a BCF of 295.80 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 267.00 L/kg wet-wt and the higher trophic level gives a value of 190.90 L/kg wet-wt.

Reference 3

Endpoint:

bioaccumulation: aquatic / sediment

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

21 SEP 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR toolbox v4.4.1

2. MODEL (incl. version number)
BCFBAF v3.01

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)c1cccc(C(C)C)c1N

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please refer to the QMRF attached under 'Attached justification'.

5. APPLICABILITY DOMAIN
Please refer to the QPRF attached under 'Attached justification'.

Guideline:

other: REACH guidance on QSAR R.6, May 2008

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR toolbox v4.4.1, EPI Suite v4.11
- Model(s) used: BCFBAF v3.01
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

Specific details on test material used for the study:

CC(C)c1cccc(C(C)C)c1N

Type:

BCF

Value:

58.23 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Regression-based estimate (Traditional method)

Type:

BCF

Value:

69.94 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas method, based on mechanistic first principles

Conclusions:

The estimation of bioconcentration factor was using the US-EPA software BCFBAFWIN v3.01, whereby using the regression-based estimate (traditional method) a BCF of 58.23 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 69.94 L/kg wet-wt.

Executive summary:

DIPA is the relevant hydrolytical degradation product of Carbodiimide (CDI). The prediction for the bioconcentration factor (BCF) of the substance 2,6-diisopropylaniline (DIPA) was determined by the computer program BCFBAFWIN v3.01 (EPIWIN software) by US-EPA. Furthermore the whole body primary biotransformation rate estimation for fish was calculated with the notation that the bio half-life normalized to 10 g fish at 15 °C. It is possible to predict the apparent metabolism half-life in fish for three different trophic levels (lower, mid and upper). Using the regression-based estimate (traditional method) a BCF of 58.23 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 69.94 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 0.301 days, whereby the rate constant (kM) for 10 g fish is designated as 0.4093/day. This is taken into account to predict the apparent metabolism half-life in fish for the substance. For the lower trophic level a BCF of 61.51 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 64.68 L/kg wet-wt and the higher trophic level gives a value of 69.94 L/kg wet-wt.

Description of key information

Key_Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) conducted a guideline study (OECD 305 -III) by exposing rainbow trout (Oncorhynchus mykiss) via diet over a 14-Day uptake period followed by a 21-Day depuration period in a flow-through limit test with the nominal concentration of 10 mg test substance/kg feed.

As radioactive material was used in this study, it was possible to evaluate the obtained results based on the analytically determined concentrations of the parent compound, the complete extractable fraction (ERR) and, as an absolute worst case scenario, the total radioactive residues (TRR), resulting from the sum of the ERR and NER. Following the worst case assumption on TRR, a BMF_KGLof 0.479 and an estimated BCF of 14631.8 resulted as key values.

Supporting_As supporting information the BCF was calculated by the QSAR model BCFBAFWIN v3.01 implemented in EPIWIN v4.1. Using the regression-based estimate (traditional method) a BCF of 1912 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 1209 L/kg wet-wt.

Supporting_As second supporting information the BCF was calculated for DIPA as relevant hydrolytical degradation product of the registered substance by the QSAR model BCFBAFWIN v3.01 implemented in EPIWIN v4.1. Using the regression-based estimate (traditional method) a BCF of 58.23 L/kg wet-wt was calculated. Using the Arnot-Gobas method, the BCF results in a value of 69.94 L/kg wet-wt.

Key value for chemical safety assessment

BCF (aquatic species):

14 631.8 dimensionless

BMF in fish (dimensionless):

0.479

Additional information

A GLP study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the biomagnification factor of the registered substance in the fish species Oncorhynchus mykiss.

 

The registered substance was administered to a test population of fish via feed in accordance to the conditions of the OECD TG 305 for BMF studies.

 

The suitability of aqueous exposure of the test item to be applied in a BCF study according to OECD TG 305 was tested and evaluated in the test facility of Fraunhofer IME (see statement ‘Suitability of an aqueous exposure for a BCF study with Bis(2,6-diisopropylphenyl)carbodiimide (non-GLP pre-test)’ dated on March 27, 2019 as attached background material). The results on the low stability of the test item in the aqueous phase provided clear indications that an aqueous exposure is not practicable.

According to OECD TG 305, a dietary exposure test can alternatively be used for substances with high hydrophobicity (test item log K_OW= 6.2) and low water solubilities (test item solubility < 0.53 µg/L). Thus, the dietary approach, generating a biomagnification factor (BMF) was further investigated to evaluate of the bioaccumulation potential of carbodiimide.

 

A test concentration of 8.02 ± 1.13 mg/kg CDI was achieved by applying solvent-mediated spiking on commercially available feed pellets. The feed pellets were pretreated by methylation of external carboxylic acid functionalities.

 

In addition, a control population of fish was fed with similar prepared feed (solvent-spiked) without the test substance to monitor potential mortalities or other adverse effects over time. Both feed types were administered with a rate of 2 % mean body weight per day.

 

The test duration was 14 days for the uptake and another 21 days for the depuration phase. No adverse effects were observed during the time course of the study.

 

Repeated samplings were done for both fish populations and five individuals were picked at each time point for chemical analyses. During the uptake phase, fish were sampled twice, at days 12 and 14 and the highest mean CDI concentration in fish was 0.322 ± 0.073 mg/kg at day 14.

 

During depuration the fish were sampled six times, at day 1, 3, 7, 10, 14 and 21 day of depuration. The test item concentration at the sampling points was used to assess the elimination kinetics of CDI in fish matrix. Based on that, a depuration rate constant of k₂ = 0.0814 was calculated.

 

With that a substance specific half-life (t_1/2) of 8.52 days could be determined.

 

The test item concentration on feed, k₂ and a test item concentration of c_0,d = 0.213 mg/kg in fish at the beginning of the depuration phase was used to determine an assimilation efficiency (α) of 0.159.

 

Based on the assimilation efficiency and the feed ingestion rate of 0.02 a kinetic BMF_K of 0.0391, a growth-corrected BMF_Kg of 0.0634 and a growth- and lipid-corrected BMF_KgL of 0.194 was specified for the registered substance.

 

As radioactive material was used in this study, it was possible to gain a broader insight into the bioaccumulating potential of test item related residues. Thus, two additional perspectives were evaluated. Notably, those evaluations constitute worst case considerations and could only be made due to the usage of radiolabeled material. The first consideration is based on the complete extractable fraction (ERR, no parent specific analysis) and the second, as an absolute worst case scenario, is based on total radioactive residues (TRR), resulting from the sum of the ERR and NER.

 

For ERR and TRR mean tissue concentrations of c_14d,ERR = 0.378 ±0.079 mg/kg and c_14d,TRR = 0.378 mg/kg were determined in fish matrix, respectively at the end of the uptake phase. Based on the analytical measurements during the depuration phase and the assessment of the elimination kinetics, the depuration rate constant (k₂) was determined to be 0.0774 for ERR and 0.0663 for TRR with substance specific half-lifes(t_1/2) of 8.96 and 10.5 days, respectively.

 

Accordingly, the assimilation efficiency was determined to be 0.159 for ERR and 0.274 for TRR, resulting in final growth - and lipid-corrected BMF_KgL of 0.251 for ERR and 0.479 for TRR.

 

By the use of the spreadsheet provided in conjunction with the Guidance Document to OECD TG 305, a BCF was estimated for the registered substance as well as for the TRR as worst-case assumption. Taking into account that the registered substance is not molecularly large containing two not halogenated aromatic rings only, the logKow was experimentally determined to be 6.2 and the assimilation efficiency within the experimental bioaccumulation study was determined to be 0.159 for the parent compound, the test substance falls within the applicability domain of the first and second BCF estimation method as outlined in the respective guidance document to OECD TG 305 (Table 4 -14). The result obtained by estimation method 3 is considered to be not reliable accordingly, as the test substance is not within its indicative applicability domains.

 

Hence, the results obtained by estimation method 1 and 2 are considered most meaningful with a worst-case BCF of 10251.0 for the parent compound (range 1020.1 - 10251.0) and 14631.8 for TRR (range 1456.0 - 14631.8). Thus, it can finally be concluded that the registered substance has a very bioaccumulative potential in the aquatic environment.

 

Modelling of the BCF was performed for the registered substance and also for the relevant hydrolytical degradation product 2,6 -diisopropylaniline (DIPA):

The prediction for the bioconcentration factor (BCF) of the registered substance was determined by the computer program BCFBAFWIN v3.01 (EPIWIN software) of US-EPA. Furthermore the whole body primary biotransformation rate estimation for fish was calculated with the notation that the bio half-life normalized to 10 g fish at 15 °C. It is possible to predict the apparent metabolism half-life in fish for three different trophic levels (lower, mid and upper). Using the regression-based estimate (traditional method) a BCF of 1912 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 1209 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 59.9 days, whereby the rate constant (kM) for 10 g fish is designated as 0.01158/day. This is taken into account to predict the apparent metabolism half-life in fish for the substance. For the lower trophic level a BCF of 295.80 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 267.00 L/kg wet-wt and the higher trophic level gives a value of 190.90 L/kg wet-wt.

No GLP criteria are applicable for the usage of this tool, but due to the fact that it is a scientifically accepted calculation method the estimations performed are reliable with restrictions and can be used for the chemical safety assessment.

 

DIPA is the relevant hydrolytical degradation product of Carbodiimide (CDI). The prediction for the bioconcentration factor (BCF) of the substance 2,6-diisopropylaniline (DIPA) was determined also by BCFBAFWIN v3.01. Using the regression-based estimate (traditional method) a BCF of 58.23 L/kg wet-wt was calculated. Using the Arnot-Gobas method, the BCF results in a value of 69.94 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 0.301 days, whereby the rate constant (kM) for 10 g fish is designated as 0.4093/day. For the lower trophic level a BCF of 61.51 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 64.68 L/kg wet-wt and the higher trophic level gives a value of 69.94 L/kg wet-wt.

 

Conclusion:

It can be concluded that relevant bioaccumulation of the hydrolysis product DIPA is not to be expected with regard to the results of the QSAR analysis but a very bioaccumulative potential has to be considered for the registered substance following the results of the experimental study according to OECD 305.