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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:
study ongoing
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:
2012
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
yes
Details on sampling:
- Sampling intervals/frequency for test organisms: Fish samples will be taken for chemical at day 0, 12 and 14 of the uptake phase as well as on day 1, 3, 7, 10, 14 and 21 of the depuration phase. For lipid analysis additional samples will be taken at day 0 and 14 of the uptake phase as well as on day 21 of the depuration phase.
- Sampling intervals/frequency for test medium samples: Feed samples for chemical and lipid analysis will be taken on day 0 and 14 of the uptake phase.
- Sample storage conditions before analysis: All samples will be immediately frozen in liquid nitrogen and stored at <-18°C.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Fish samples for chemical analysis: At each sampling event five fish from the treatment and the control vessel will be removed, immediately rinsed in dilution water, blotted dry and will then be weighed and measured before being humanely killed. Each fish will be analyzed individually for the test item to generate five replicate data per sampling date. For the controls, the analysis of only one replicate sampled at the beginning (from the stock population) and end of the exposure phase, as well as at the end of the depuration phase will be sufficient in case of obvious absence of background contamination. No additional fish will be sampled from the stock population for chemical analysis during the study, as the fish sampled from the control chambers can be used to determine potential background levels of the test item.
Total radioactive residues (TRR), extractable radioactive residues (ERR) and non-extractable radioactive residues (NER) of the post-extraction solid will be determined. Parent-specific analysis will be performed by radio-thin layer chromatography (TLC) analysis of the ERR.
Fish samples for lipid analysis: Fish will be sampled in triplicates from the treatment and the control at the end of the uptake period as well as the end of the depuration period for lipid analysis. Three additional reference fish will be sampled from the stock population at the beginning of the test. All lipid samples will be analysed for total lipid content according to a method by Smedes.
Feed samples for chemical analysis: Triplicate samples of test and control diet will be taken for chemical analysis. The test item on fish feed will be analysed using liquid chromatography with tandem mass spectrometry (LC-MS/MS).
Feed samples for lipid analysis: Triplicate samples of test and control diet will be analysed for the lipid content. All lipid samples will be analysed for total lipid content according to a method by Smedes.
Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): Commercially available trout feed pellets.
- 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 feed will be spiked with the test substance at one concentration by solvent spiking. Before spiking, potentially reactive groups on the feed pellet surface (e.g. carboxylic acids) will get masked through methylation to suppress possible covalent reactions of reactive functionalities of feed pellets with the test substance.
For the purpose of methylation, 1 % sulfuric acid in methanol (v/v) will be spread onto the feed pellets via spray application. For spray application, a rotary evaporator will be connected to a stainless steel capillary solvent-inlet tube to obtain a simple vacuum spiking apparatus. A 2 L pear-shaped drying flask will be used as rotating flask for spray application and filled with an appropriate amount of test feed (e.g. 100 g). During the application, the pressure of the vacuum system will be set to approximately 600 mbar and the rotational speed will be set to sufficiently reach an even distribution of the pellets on the inner surface of the flask. The flask will be kept in a water bath not warmer than 40 °C. After the application of the sulfuric acid, the capillary inlet tube will be sealed with parafilm and the pressure will be reduced to approximately 400 mbar for another few minutes (e.g. 10-20 min) of rotation to ensure complete evaporation of the solvent. Thereafter the pretreated feed batch will be incubated in a drying chamber at 55 °C overnight.
For application of the test item, an appropriate amount of the test substance will be dissolved in an appropriate amount of solvent (e.g. acetone) and the test item will be applied via spray application as described above. Afterwards, the spiked pellets will be divided in appropriate portions for daily feeding and stored at ≤ -18 °C until use. The control diet will be prepared exactly the same way as the spiked diet, but without test substance in the spiking solvent.
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 fish of s
- Length at study initiation (length definition, mean, range and SD): s imilar weight and length (8 ± 4 cm)
- Weight at study initiation (mean and range, SD): similar weight and length (8 ± 4 cm)
- Weight at termination (mean and range, SD): information not available yet
- Method of breeding: The fish will be held in water of the same quality as used in the test (purified drinking water) until the start of exposure.
- Lipid content at test initiation (mean and range, SD): information not available yet
- Health status: Only healthy fish, free from observable diseases and abnormalities will be used in the study. Mortality of the batch must be 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 will be held under equivalent water quality and illumination conditions to those proposed for use in the test.
- Type and amount of food: Fish will be fed ad libitum throughout the acclimation period with trout feed.
- Feeding frequency: once daily
- Health during acclimation (any mortality observed): information not available yet
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:
information not available yet
Test temperature:
The temperature of holding and dilution water during the test will be maintained at 15 ± 2 °C.
pH:
information not available yet
Dissolved oxygen:
The dissolved oxygen concentration will be kept above 60 % ASV (Air Saturation Value) throughout the test.
TOC:
information not available yet
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 will be aerated via a glass capillary.
- 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 will be 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: fish-to-water loading rate of 0.1 to 1.0 g fish (wet weight) per liter of water per day will be applied

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-chlorinated local tap water will be used in accordance with the OECD guideline. The tap water is sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process occurs on-site at Fraunhofer IME and includes filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation. To avoid copper contamination, plastic water pipes are used in the test facilities.
- Holding medium different from test medium: no
- Intervals of water quality measurement: 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] recorded monthly in the laboratory of the test facility will be reported.

OTHER TEST CONDITIONS
- Adjustment of pH: information not available yet
- 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): information not available yet
- 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: information not available yet
- 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: information not available yet

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 1 and 10 mg/kg
Nominal and measured concentrations:
nominal: 10 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)
Remarks on result:
other: will be available, once study is finished
Remarks on result:
other: will be available, once study is finished
Validity criteria fulfilled:
not applicable
Remarks:
study ongoing
Conclusions:
A GLP study according to OECD TG 305-III: Dietary Exposure Bioaccumulation Fish Test is currently running at Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the dietary biomagnification factor (BMF) of Bis(2,6-diisopropylphenyl)carbodiimide (14C-Carbodiimide; CDI). A Statement by Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) dated on December 03, 2020 is attached as background material, summarising the reasons for the delay of the performance of the definitive test. Accordingly the study could not yet been completed, wherefore it is not possible to draw a conclusion on the bioaccumulation potential of the registered substance at current stage.
Executive summary:

A GLP study according to OECD TG 305-III: Dietary Exposure Bioaccumulation Fish Test is currently running and ongoing at Fraunhofer GmbH to determine the dietary biomagnification factor (BMF) of Bis(2,6-diisopropylphenyl)carbodiimide (14C-Carbodiimide; CDI).

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 KOW= 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.

The test consists of two phases: uptake (test substance-spiked feed) and depuration (clean, untreated feed). In the uptake phase, a “test” group of fish is fed a fixed amount of diet (commercial fish feed with a known composition) spiked with the test substance, on a daily basis. The fish ideally must consume all of the offered food. During the depuration phase fish are fed with pure, untreated commercial fish feed.

The concentration of the test substance in fish is measured during both phases of the test. In addition to the group of fish fed the spiked diet (the test group), a control group of fish is held under identical conditions and fed the same amounts except that the commercial fish feed is solvent-spiked only. This control group will be assessed for background levels of test substance in fish matrix and serves as a comparison for any treatment-related adverse effects noted in the test group. It also allows a comparison of growth rate constants between the groups.

An uptake phase that lasts 7-14 days is generally sufficient. During the uptake phase the chemical concentration in the fish may not reach steady-state so data treatment and results from this method are usually based on a kinetic analysis of tissue residues during depuration. The depuration phase begins when the fish are first fed unspiked diet and typically lasts for up to 28 days or until the test substance can no longer be quantified in the fish, whichever is the sooner.

Lipid content of sampled whole fish should be measured so that concentrations can be lipid-corrected, taking account of lipid content of both, the diet and the fish.

This method allows the determination of the following parameters for the test substance in fish:

• substance-specific half-life (t1/2, from the depuration rate constant, k2)

• assimilation efficiency (absorption across the gut;α)

• growth rate constant (day-1, kg)

• growth corrected depuration rate constant (day-1, k2g)

• kinetic dietary biomagnification factor (BMFk)

• growth-corrected kinetic dietary biomagnification factor (BMFkg)

• and/or growth- and lipid-corrected kinetic dietary biomagnification factor (BMFkgL)

A Statement by Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) dated on December 03, 2020 is attached as background material, summarising the reasons for the delay of the performance of the definitive test and an estimated scheduling for further procedure.

Description of key information

A GLP study according to OECD TG 305-III: Dietary Exposure Bioaccumulation Fish Test is currently running and ongoing at Fraunhofer GmbH to determine the dietary biomagnification factor (BMF) of Bis(2,6-diisopropylphenyl)carbodiimide (14C-Carbodiimide; CDI).

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 KOW= 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 the bioaccumulation potential of carbodiimide.

To establish a stable enriched diet for experimentation, developmental work for the GLP study entitled ‘Dietary exposure bioaccumulation fish test with rainbow trout (Oncorhynchus mykiss)’has been completed during June 2019 and February 2020. Due to its reactive character, the application of the test item is difficult. Different tests and approaches were performed to ensure that the protocols for the production and storage of enriched feed for the test suffice with regard to the test items stability. This work was summarized in ‘Development of an dietary exposure for a BMF study with Bis(2,6-diisopropylphenyl)carbodiimide’dated onFebruary 05, 2020 and is attached as background material for complete information.

Findings from the method development for test diet raised the concern of the formation of covalent bonds between the test item and molecules inside the organism what may lead to too low parent concentrations to determine relevant data. To facilitate the analysis of the test item on feed and in fish, it was decided to continue the testing with radiolabelled material.

The test itself consists of two phases: uptake (test substance-spiked feed) and depuration (clean, untreated feed). In the uptake phase, a “test” group of fish is fed a fixed amount of diet (commercial fish feed with a known composition) spiked with the test substance, on a daily basis. The fish ideally must consume all of the offered food. During the depuration phase fish are fed with pure, untreated commercial fish feed.

The concentration of the test substance in fish is measured during both phases of the test. In addition to the group of fish fed the spiked diet (the test group), a control group of fish is held under identical conditions and fed the same amounts except that the commercial fish feed is solvent-spiked only. This control group will be assessed for background levels of test substance in fish matrix and serves as a comparison for any treatment-related adverse effects noted in the test group. It also allows a comparison of growth rate constants between the groups.

An uptake phase that lasts 7-14 days is generally sufficient. During the uptake phase the chemical concentration in the fish may not reach steady-state so data treatment and results from this method are usually based on a kinetic analysis of tissue residues during depuration. The depuration phase begins when the fish are first fed unspiked diet and typically lasts for up to 28 days or until the test substance can no longer be quantified in the fish, whichever is the sooner.

Lipid content of sampled whole fish should be measured so that concentrations can be lipid-corrected, taking account of lipid content of both, the diet and the fish.

This method allows the determination of the following parameters for the test substance in fish:

• substance-specific half-life (t1/2, from the depuration rate constant, k2)

• assimilation efficiency (absorption across the gut;α)

• growth rate constant (day-1, kg)

• growth corrected depuration rate constant (day-1, k2g)

• kinetic dietary biomagnification factor (BMFk)

• growth-corrected kinetic dietary biomagnification factor (BMFkg)

• and/or growth- and lipid-corrected kinetic dietary biomagnification factor (BMFkgL)

A Statement by Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) dated on December 03, 2020 is attached as background material, summarising the reasons for the delay of the performance of the definitive test and an estimated scheduling for further procedure.

Key value for chemical safety assessment

Additional information

The above mentioned bioaccumulation study is currently performed as requested in the final decision of ECHA on a compliance check, which was issued 01 August 2017. Due to difficulties in the setup of the study, it is still under progress.

Because new data is generated at this time, the BCF value is under revision and the previous data generated via QSAR is regarded as supporting information only.

Modelling of the BCF was performed for bis(2,6 -diisopropylphenyl)carbodiimide and also for the relevant hydrolytical degradation product 2,6 -diisopropylaniline (DIPA).

The prediction for the bioconcentration factor (BCF) of the substance bis(2,6-diisopropylphenyl)carbodiimide was determined by the computer program BCFBAFWIN v3.01 (EPIWIN software) of US-EPA (Chemservice S.A., 2011). 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 (Chemservice S.A., 2016). 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 carbodiimide and of the hydrolysis product DIPA is not to be expected. Regarding environmental fate, DIPA should be assessed, since Carbodiimide is hydrolytically unstable.