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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
July 26, 2013
Deviations:
yes
Remarks:
Analytical confirmation of the test concentration was not performed as the test item is an organic UVCB substance showing fast degradation in water, e.g. by hydrolysis.The dissolved organic carbon was measured to act as replacement of a specific analysis.
Principles of method if other than guideline:
Specific analytical confirmation of the test concentration was not performed due to technical reasons. The test item is an organic UVCB substance showing fast degradation in water, e.g. by hydrolysis. As the potential ecotoxicity of the test item cannot be assigned to a specific constituent, no appropriate analytical method for an accurate analysis can be developed. The dissolved organic carbon (DOC) was measured to act as replacement of a specific analysis of the test item.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
The dissolved organic carbon (DOC) was measured to act as replacement of a specific analysis of the test item.
Details on sampling:
SAMPLING SCHEDULE
-Samples were taken from the storage tanks providing the test solutions (e.g. storage tank of control medium and/or treatment level (filtrate)).
-As the dissolved organic carbon (DOC) was measured to act as replacement of a specific analysis of the test item in the test media the samples for chemical analysis could consequently not be taken from the test vessels containing additional organic carbon sources (e.g. faeces of fish, food).
-Samples of the control were taken at three occasions during the study, at start of the test (day 0), once in week 3 and at end of the test (day 34).
-Samples of each freshly prepared stock solution(s) were taken immediately before use, samples were taken once a day.
-Samples of aged stock solution were taken from the storage tank providing the stock solution (e.g. storage tank), samples were taken once a day.

STORAGE CONDITIONS
Samples were stored deep frozen and protected from light until they were analysed.
Vehicle:
no
Details on test solutions:
PREPARATION OF STOCK SOLUTION
Stock solutions (S1) of the test item in modified reconstituted water were prepared by weighing 0.2017 – 0.25235 g of the test item into 5 L of dilution medium, resulting in nominal concentrations of 40 – 50 mg test item/L (August 27, 2018 to September 30, 2018). This solution (5 L) was treated by ultrasonication for 1 hour to ensure a homogeneous distribution of the test item in the test medium.
Thereafter stock solution (S1), containing the pre-dissolved test item, was combined with the total amount of test medium used to prepare the final stock solution (S2). Therefore, the stock solution (S1) was transferred in a stainless steel tank. The measuring flask containing the stock solution S1 was rinsed once with modified reconstituted water (filled up with test medium (up to calibration mark)) and added to the storage tank of the final stock solution (S2). The remaining amount of modified reconstituted water was added to the stock solution tank using 5 and 10 L measuring flasks, if necessary, so that in total 20 – 25 L (depending on the final volume of the stock solution S2) of modified reconstituted water was provided in the stainless steel tank.
This final stock solution was stirred for 24±3 hours at ambient temperature in the dark using a stainless steel propeller stirrer. Subsequently the suspension was filtered, using a glass fibre filter with an average retention capacity of 0.4 µm (Macherey-Nagel, MN85-220). The filtrate was visually examined for un-dissolved/particulate matter (e.g. using the Tyndall effect, microscopic control). The filtrate was considered the stock solution (S2) and was used to produce the desired test item concentration on the day of filtration. The filtrate was stored at room temperature in the dark.

PREPARATION OF TEST SOLUTIONS
-The control medium and the stock solution (test solution) was delivered directly from the storage vessel to the designated test vessels using teflon tubing and a multi-channel peristaltic pump (Tygon tubes), set to deliver the desired volume.
-During dosing into the flow-through system (between renewals), the control and the test item solutions were stored at room temperature in the dark. During storage, the tanks were closed with a lid to reduce air-exchange and evaporation.
-The flow rates (volume delivered per time) were measured volumetrically three times per week. The volume delivered was divided by the time. The variation of the nominal volume setting (2.78 mL/min, equivalent to 4000 mL/day) was calculated by dividing the measured volume per time [mL/min] by the nominal volume setting of the designated pump expressed in percentage of nominal values. Additionally the distribution between the replicates was measured three times per week.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Species: Danio rerio Hamilton-Buchanan 1822 (Zebrafish)
- Source: Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since June 2017

HOLDING CONDITIONS AND FERTILISATION OF TEST ORGANISMS:
In the culture tanks, the fish were kept in reconstituted water (OECD guideline 203, 1992) diluted with deionised water (1:1; v/v) and supplemented with 1 % of artificial seawater (Tropic Marin, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28 ± 2 ‰). Fish were held in glass aquaria at 26 ± 2 °C with a 14 h/10 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. (Sanders Brine Shrimp Co., Morgan, UT 84050, U.S.A.) and TetraMin (Tetra Werke, Melle, Germany).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding aquaria of the adult zebrafish. Water plants were placed on the mesh, allowing the fish to spawn.
-The spawning bowls were removed from the holding aquaria on the day of test start shortly after onset of illumination (i.e. 60 minutes), which triggers fertilization. The content of the bowls was poured over a sieve, rinsed with tempered reconstituted water and collected in a glass vessel filled with tempered reconstituted water. Immediately afterwards, groups of eggs were transferred to glass dishes (one replicate per test concentration) containing test solution of each designated exposure vessel, acclimated at test temperature. After each pre-exposure vessel contained about 250 eggs, the dishes were placed into an incubator set to 26 °C (26 ± 1.5 °C) for 1 hours and 10 minutes. Thereafter, groups of fertilised eggs were transferred to an additional set of pre-exposure vessels (six replicates per test concentration) containing 50 mL of temperature-adapted test solutions using a randomisation procedure. This was done by sequentially adding groups of fertilised eggs (5 eggs per test vessel) from the first pre-exposure vessel for each test concentration into the corresponding replicated pre-exposure vessels until each test vessel contained 20 eggs. Thereafter the fertilised eggs (20 per pre-exposure vessel) were transferred into the test units.

FEEDIND
On day 3 of exposure, after the first larva per test vessel was recorded to be hatched, feeding was started by adding food to the vessels. The test organisms were fed on dry food (Novo Baby 01 and Novo Baby 02, JBL GmbH & Co. KG, Neuhofen, Germany), live Artemia nauplius larvae and paramaecia (Paramecium caudatum) ad libitum. The daily ration was fed in 2 - 4 equal portions each day, except on day 3 were the fish was fed in one portion. Uneaten food was removed once daily from day 5 of exposure. The food ration was adjusted to the number of living fish per test vessel. Food was withheld from the fish for 24 hours prior to test end.
Test type:
flow-through
Water media type:
freshwater
Remarks:
reconstituted water, diluted with deionised water (1:1, v/v) and supplemented with 1 % of artificial seawater
Limit test:
yes
Total exposure duration:
34 d
Hardness:
9.2-10.0 dH (164-179 mg CaCO3/L)
Test temperature:
26 ± 1.5 °C
pH:
7.1-7.6
Dissolved oxygen:
5.2-8.0 mg/L (66-99 %)
Salinity:
n.a.
Conductivity:
762-807 µS/cm
Nominal and measured concentrations:
10 mg/L nominal concentration
Details on test conditions:
TEST SYSTEM:
- Glass vessels of appropriate sizes (1000 mL volume)
- Dimensions of test vessels: Diameter: 14.0 cm, Height: 7.5 cm; vessels were fitted with a sideward fixed meshed overflow
- Volume of test solution per test vessel: 0.8 L (nominal)
- Test vessels covered with a transparent cover

EXPOSURE CONDITIONS:
- Photoperiod: light/dark cycle 14 h/10 h.
- Light intensity: measured: 595 - 686 lx.
- Temperature: 26 ± 1.5 °C (target); measured min/max: 26.0/26.6 °C (manual); 25.5/26.3 °C (automatic).
- Aeration: the test vessels were gently aerated during the test using teflon tubes.
- Renewal of the test solution during the test period: flow-through, five vessel volumes per vessel and day.
- The flow rate was adjusted so that the loading (biomass per volume of test solution) did not exceed a loading of 5 g fish/L of solution and a loading rate of 0.5 g fish/L per 24 hours at any time of the test (see below).
- To calculate the maximum loading, the maximum total fish wet weight per vessel (1.0513 g in the controls, replicate c) was divided by the vessel volume (0.8 L). The maximum loading was 1.31 g fish/L.
- The loading rate was calculated by dividing the maximum total fish wet weight per vessel (1.0513 g in the controls, replicate c) by the target flow rate of five vessel volumes per day (4.0 L/vessel/day). The maximum loading rate was 0.263 g fish/L per 24 hours.
- Feeding: On day 3 of exposure, after the first larva per test vessel was recorded to be hatched, feeding was started by adding food to the vessels. The test organisms were fed on dry food (Novo Baby 01 and Novo Baby 02, JBL GmbH & Co. KG, Neuhofen, Germany), live Artemia nauplius larvae and paramaecia (Paramecium caudatum) ad libitum. The daily ration was fed in 2 - 4 equal portions each day, except on day 3 were the fish was fed in one portion. Uneaten food was removed once daily from day 5 of exposure. The food ration was adjusted to the number of living fish per test vessel. Food was withheld from the fish for 24 hours prior to test end.
- Dead eggs, embryos or fish were removed from the test vessels upon daily observation throughout the test.
- The flow-through system including test vessels were conditioned with the test solutions for one day prior to start of exposure
- Note (conditioning phase): The environmental conditions (temperature, light) during the conditioning phase were:
Temperature: 26±1.5 °C (target),
Photoperiod: light/dark cycle 14 h/10 h,
Light intensity: comparable to the exposure phase,
Aeration: the test vessels were aerated,
Renewal of the test solution during the conditioning phase: flow-through, five vessel volumes per vessel and day.
Reference substance (positive control):
no
Key result
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.033 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
based on mean measured DOC concentrations in the test solutions given in mg DOC/L
Basis for effect:
other: Hatching success, Post-hatch success (survival), number of healthy fish, length of the surviving fish, wet weight of the surviving fish, dry weight of the surviving fish
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 0.033 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
based on mean measured DOC concentrations in the test solutions given in mg DOC/L
Basis for effect:
other: Hatching success, Post-hatch success (survival), number of healthy fish, length of the surviving fish, wet weight of the surviving fish, dry weight of the surviving fish
Duration:
34 d
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Basis for effect:
other: Hatching success, Post-hatch success (survival), number of healthy fish, length of the surviving fish, wet weight of the surviving fish, dry weight of the surviving fish
Duration:
34 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Basis for effect:
other: Hatching success, Post-hatch success (survival), number of healthy fish, length of the surviving fish, wet weight of the surviving fish, dry weight of the surviving fish
Details on results:
The measured concentrations in the stock solutions range between 0.0 and 0.385 mg DOC/L in freshly prepared stock solutions (filtrate) and between 0.0 and 00.287 mg DOC/L in aged stock solutions (filtrate). In all cases the concentration measured in the freshly prepared stock solutions (Sf) were higher than in the aged stock solutions. To calculate the overall mean concentration, the geometric mean concentration of each corresponding data set (fresh and aged stock solution of consecutive days) were calculated and from these geometric mean concentrations the arithmetic mean overall concentration was calculated. The overall mean measured concentration was calculated to be 0.033 mg DOC/L.
The biological endpoints (NOEC/LOEC) were reported based on mean measured DOC concentrations in the test solutions given in mg DOC/L, corresponding measured test item concentration in test solutions given in mg test item/L.

SUMMARY OF BIOLOGICAL RESULTS

At test end, in the controls 68 healthy fish from 80 introduced eggs survived. In the test item treatment group the number of living, healthy fish was 64.

 Nominal concentration [%]*  Introduced eggs [n]  Hatched fish [n]  Survived fish [n]  Deformed fish/different behaviour [n]  Healthy fish [n] Wet weight of fish [mg]  Dry weight of fish [mg]  Length of fish [cm] 
 0 (Control)  80  77  68  1  67  56.09  14.65  1.958
 100  80  77  64  0  64  56.77  14.72  1.979

*% of a filtrate at a loading rate of 10 mg test item/L

Validity criteria fulfilled:
yes
Conclusions:
No chronic toxic effect of 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type to Danio renio at a limit concentration of 10 mg/L (nominal) could be observed.
Executive summary:

The study was conducted according to OECD Guideline for Testing of Chemicals, No. 210 "Fish, Early-life Stage Toxicity Test" to determine lethal and sub-lethal effects of 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type on the early-life stages of Danio rerio (zebrafish). To achieve this, a limit concentration of the test item in aqueous solution was prepared using a filtrate at a loading rate of 10 mg test item/L. The test organisms were exposed to this limit concentration as well as to control without the test item. During the test period the test solutions were renewed in a flow-through system.

Newly fertilised eggs of the test species were exposed to this test concentration until 30 days post-hatch. Lethal and sub-lethal effects of the test item on eggs, larvae and juvenile fish were assessed in comparison with control values to determine the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC). The following biological parameters were investigated: hatching success; number of larvae hatching each day; time to start of hatching and end of hatching; cumulative mortality, post-hatch success (survival); number of deformed or abnormally appearing larvae; number of organisms exhibiting abnormal behaviour; length of the surviving fish; wet and dry weight of the surviving fish. 6 replicates of 20 fertilized eggs per concentration and control were exposed.

To determine the actual test item concentrations, samples were taken from the stock solution for analytical measurement. Samples were measured in stock solution at regular intervals throughout the exposure period.

Deviating from the test guideline: specific analytical confirmation of the test concentration was not performed due to technical reasons. The test item is an organic UVCB substance showing fast degradation in water, e.g. by hydrolysis. As the potential ecotoxicity of the test item cannot be assigned to a specific constituent, no appropriate analytical method for an accurate analysis can be developed. The dissolved organic carbon (DOC) was measured to act as replacement of a specific analysis of the test item.

The measured DOC concentrations in the freshly prepared stock solutions range between 0.0 and 0.385 mg DOC/L and between 0.0 and 0.287 mg DOC/L in the aged stock solutions. The mean measured DOC concentration in test solution was calculated to be 0.033  mg DOC/L.

The biological endpoints (NOEC/LOEC) were reported based on mean measured DOC concentrations in the test solutions given in mg DOC/L, corresponding measured test item concentration in test solutions given in mg test item/L.

As no chronic toxic effects were observed, a NOEC of ≥10 mg/L (nominal) equaling ≥0.033 mg/L (mean measured) and a LOEC of >10 mg/L (nominal) equaling >0.033 mg/L (mean measured) were determined for 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type for hatching success, post-hatch success (survival), number of helthy fish, length of surving fish, wet and dry weight of surviving fish, respectively.

Description of key information

As no chronic toxic effects were observed, a NOEC of ≥10 mg/L (nominal) equalling ≥0.033 mg/L (mean measured) and a LOEC of >10 mg/L (nominal) equalling >0.033 mg/L (mean measured) were determined for 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type for hatching success, post-hatch success (survival), number of healthy fish, length of surviving fish, wet and dry weight of surviving fish, respectively.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
10 mg/L

Additional information

Result should read as ≥10 mg/L (nominal)

The study was conducted according to OECD Guideline for Testing of Chemicals, No. 210 "Fish, Early-life Stage Toxicity Test" to determine lethal and sub-lethal effects of 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type on the early-life stages of Danio rerio (zebrafish). To achieve this, a limit concentration of the test item in aqueous solution was prepared using a filtrate at a loading rate of 10 mg test item/L. The test organisms were exposed to this limit concentration as well as to control without the test item. During the test period the test solutions were renewed in a flow-through system.

Newly fertilised eggs of the test species were exposed to this test concentration until 30 days post-hatch. Lethal and sub-lethal effects of the test item on eggs, larvae and juvenile fish were assessed in comparison with control values to determine the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC). The following biological parameters were investigated: hatching success; number of larvae hatching each day; time to start of hatching and end of hatching; cumulative mortality, post-hatch success (survival); number of deformed or abnormally appearing larvae; number of organisms exhibiting abnormal behaviour; length of the surviving fish; wet and dry weight of the surviving fish. 6 replicates of 20 fertilized eggs per concentration and control were exposed.

To determine the actual test item concentrations, samples were taken from the stock solution for analytical measurement. Samples were measured in stock solution at regular intervals throughout the exposure period.

Deviating from the test guideline: specific analytical confirmation of the test concentration was not performed due to technical reasons. The test item is an organic UVCB substance showing fast degradation in water, e.g. by hydrolysis. As the potential ecotoxicity of the test item cannot be assigned to a specific constituent, no appropriate analytical method for an accurate analysis can be developed. The dissolved organic carbon (DOC) was measured to act as replacement of a specific analysis of the test item.

The measured DOC concentrations in the freshly prepared stock solutions range between 0.0 and 0.385 mg DOC/L and between 0.0 and 0.287 mg DOC/L in the aged stock solutions. The mean measured DOC concentration in test solution was calculated to be 0.033 mg DOC/L. As the DOC cannot be applied to the parent substance, results are expressed in nominal values.

The biological endpoints (NOEC/LOEC) were reported based on mean measured DOC concentrations in the test solutions given in mg DOC/L, corresponding measured test item concentration in test solutions given in mg test item/L.