[No public or meaningful name is available]

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06.2021 - 09.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ECETOC. Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances [S]. ECETOC Monograph No. 26 1996.

Version / remarks:

1996

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. HJ/T 153-2004, The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press.2004.

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

other: ASTM international. Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. D6081 − 20 (Reapproved 2020).

Version / remarks:

2020

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2013

Qualifier:

according to guideline

Guideline:

other: OECD. OECD series on testing and assessment number 23 (Second Edition), Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. Paris: OECD, Adopted on February 8, 2019.

Version / remarks:

2019

Qualifier:

according to guideline

Guideline:

other: China National Technical Committee for Standardization of Dangerous Chemical Management GB/T 21854-2008.Chemical Fish, Early-life Stage Toxicity Test [S]. Beijing: China Standardization Press, 2008.

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 210 Fish, Early-life Stage Toxicity Test [M]. Second Edition. Beijing: China Environmental Science Press, 2013:86-93.

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Dechlorinated tap water was used in this test. Before the test, the test water was dechlorinated by aeration so that the dissolved oxygen concentration reached more than 80% of the air saturation value. The quality parameters measured regularly on the test water is shown in Annex A.

Test organisms (species):

other: Rare Minnow (Gobiocypris rarus)

Details on test organisms:

Test fish
(1) Species: Rare Minnow（Gobiocypris rarus）
(2) Reason for selection of species: The test species is recommended by “The Guidelines for the Testing of Chemicals, 210 Fish, Early-life Stage Toxicity Test” (Chemical Registration Center of MEP, Second Edition).
(3) Supplier: The parent fish with the batch NO. FGr20201027-1 were purchased from Institute of Hydrobiology, Chinese Academy of Science. The brood fish were hold in the water maintained the temperature at 25 °C ± 2 °C with 14 h light /10 h darkness. The female and male were paired as 1:1, the oocytes and semens were collected from the parent fish with obviously tailgating behaviours, and obtained the embryos by the method of artificial fertilisation.
(4) Selection and treatment of embryo: Embryos with normal development and good morphology were selected under an anatomical microscope. The normal division embryos are sticky and transparent. However, unfertilized embryos are observed as milky white.
(5) The size of embryo: The embryos exposed not older than 8 h after fertilized
(6) Number of embryo: 360 embryos.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Nominal and measured concentrations:

1.0 - 3.2 - 10 - 32 -100 mg/L and a control

Details on test conditions:

Test conditions
(1) Test substance LICOCARE RBW 300 FL TP
(2) Test organism Embryos of Rare Minnow (within 8 hours after fertilisation)
(3) Exposure duration 34 d
(4) Test concentration According to the results of preliminary test (see Annex C), five nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L water accommodated fraction (WAF) test groups with a geometric factor of 3.2 and one blank control were set in the definitive test.
(5) Replicate 3 replicates / test group
(6) Number of embryo 20 embryos / replicate, 60 embryos / test group, 360 embryos in total.
(7) Test water Dechlorinated tap water
(8) Type of test Renewal more than 3/4 volume test solution every 24 h by the way of semi-static test.
(9) Preparation of the test solution 1d prior to the test – 8 d: 2.0 mg, 6.4 mg, 20 mg, 64 mg and 200 mg test substance were weighed into five beakers that contained 2000 mL dechlorinated tap water, the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L test suspensions were obtained. The suspensions were stirred for about 24 h, and the stirring intensities were sufficient to maintain vortex heights of about 10% of liquid depths. After the completion of stirring, the suspensions were allowed to stand for 1 hour. Then about 600 mL test solution from the middle layer were siphoned into another beakers, the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and divided into three replicates as test solutions (200 mL / replicate).
9 d – 32 d: 10 mg, 32 mg, 100 mg, 320 mg and 1000 mg test substance were weighed into five jars that contained 10000 mL dechlorinated tap water, the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L test suspensions were obtained. The suspensions were stirred for about 24 h, and the stirring intensities were sufficient to maintain vortex heights of about 10% of liquid depths. After the completion of stirring, the suspension were allowed to stand for 1 hour. Then about 6000 mL test solution from the middle layers were siphoned into another 10L jars, the nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF solutions were obtained and divided into three replicates as test solutions (2000 mL / replicate).
Note：The above mentioned about preparation of test solution was in accordance with the OECD guidance document No. 23 (2019)
(10) Volume of test solution 0 d – 9 d: 200 mL / test vessel, 10 d- 34 d: 2000 mL / test vessel.
(11) Temperature of test solutions The temperature of all test vessels measured regularly ranged from 24.7°C to 25.1°C, the temperature of the water bath was measured continuously ranged from 24.0 °C to 25.4 °C.
(12) Photoperiod 14 h light / 10 h darkness
(13) Analysis of actual concentrations of TOC in test solutions
The actual concentrations of TOC in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were measured at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) by TOC analyzer.

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Hatching rate, abnormal behavior, surviving rate, weight, lenght

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Hatching rate

Details on results:

Results
(1) Basis of the TOC as concentration analysis
LICOCARE RBW 300 FL TP with the character of low water solubility. And it’s not soluble in regular solvent (Methanol, acetonitrile, tetrahydrofuran, isopropanol, etc), so HPLC (High Performance Liquid Chromatography) and LC-MS/MS (Liquid Chromatography-Mass Spectrometer) is not suitable for analysis. According to the GC (Gas Chromatography) method in Certificate of Analysis that sponsor provided, the test substance should be derived first, but derivation must be proceeded under non-aqua system, which is not suitable for this test system and the analysis of water solution, meanwhile, the predicted limit of quantification (LOQ) of the analysis method would be high. Moreover, the carbon chain of some component of the test substance is much more than 40, so it’s not guaranteed that all constituents of test substance would be peaked in GC condition because of the high boiling point, and it would cause residual and pollute the apparatus. Consequently, in this test, only the concentrations of TOC in test solutions were measured to indicate the stability of exposed concentration of the test system.
(2) Basis of preparation of test solution
The LICOCARE RBW 300 FL TP with the character of Light yellow flakes solid, is a complex mixture/UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media was prepared following OECD guidance document No. 23 (2019), preparation the test solution by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF solution was separated by siphon. The WAF solution was checked for any undissolved or emulsified material by the Tyndall effect. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.
(3) The measured concentrations of TOC in test solutions
The limit of detection (LOD) and the limit of quantification (LOQ) of TOC were 0.2 mg/L and 0.8 mg/L, respectively. The linearity range of this test was 0.80 mg/L to 50.0 mg/L.
The measured concentrations of TOC in blank control at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) ranged from 0.873 mg/L to 1.92 mg/L.
The measured concentrations of TOC in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) ranged from < 0.8 mg/L (LOQ) to 2.04 mg/L. The measured concentrations of TOC in all test substance groups were very similar to those of the blank control, indicating that test substance was poorly soluble in test water.
(4) Toxicity effects on test organism
During the test periods, except the dead embryos or larvae, there no abnormal appearances or behaviours were observed. Under this test condition, at the end of the test (34 d), the No Observed Effect loading rate (NOEL) and Lowest Observed Effect loading rate (LOEL) and the 10% effective loading rate of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:





Indicator EL10 NOEL LOEL
Hatching rate — =100 mg/L >100 mg/L
The abnormal rate of embryos/larvae — =100 mg/L >100 mg/L
surviving rate — =100 mg/L >100 mg/L
Total length of larvae/juvenile fish >100 mg/L =32 mg/L =100 mg/L
The individual wet weight of larvae/juvenile fish (blotted-dry) >100 mg/L =100 mg/L >100 mg/L
Dry weight of larvae/juvenile fish >100 mg/L =100 mg/L >100 mg/L

Observation and measurement

(1) Observation embryos and larvae or juvenile fish

Stage of embryonic development: The embryonic stages were recorded at the start of exposure time.

Hatching and survival: Observations on hatching and survival were made daily and numbers recorded, removed dead individuals.

Abnormal appearance: The numbers of embryos or larvae or juvenile fish showing abnormality of body form were recorded daily.

Abnormal behaviour: The behavour of larvae or juvenile fish after hatched were observed and recorded daily.

(2) Temperature, pH and dissolved oxygen concentration

The temperature, pH and dissolved oxygen of all test vessels at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 15 d, 22 d, 28 d, 34 d (old solution). Meanwhile, the temperature of incubator or water bath of the test environment was continuously monitored during the test.

(3) Total hardness of test solution

Total hardness of blank control and 100 mg/L WAF test group were measured at the beginning of the test.

(4) Salinity of test solution

The salinity of the blank control and all test groups were measured at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 15 d, 22 d, 28 d, 34 d (old solution).

(5) Measurement of individual lengths

Total lengths of surviving larvae or juvenile fish were measured at the end of the test.

(6) The individual wet weight (blotted-dry) and dry weights of larvae or juvenile fish

At the end of the test, all surviving test larvae were weighed one bye one, and all surviving fish on a replicate basis were taken into a culture dish and dried at 60 ºC for 24 hours, the culture dishes were dried at 105°C for 2 hours prior to use.

8 Results

8.1 Observation of test solutions

The WAF solution was prepared with the direct addition method. During the preparation periods, particulate matters were observed suspending in all test suspensions, so the suspension solution was allowed to stand for 1h and the test solution in the middle layer was siphoned into another test vessel as exposure solution, certainly, there no obvious insoluble matters were observed in the exposure solution by the observed method of Tyndall effect.

8.2 The temperature, pH, dissolved oxygen, hardness and salinity

The results of pH, dissolved oxygen and temperature of new test solution at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d and old solution at 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d are shown in Table 1- 4 and Figure 1.

During the test, the pH of the test solution was ranged from 7.61 to 8.32. The temperature of all test vessels measured regularly ranged from 24.7°C to 25.1°C, and the test environment of water bath was measured continuously ranged from 24.0 °C to 25.4 °C, the temperature was not differ by more than ±1.5 °C between test chambers or between successive days at any time during the test, and the temperature was within the range of 25°C ± 1°C. The dissolved oxygen concentration ranged from 61% to 99% of the air saturation value (ASV) and met the requirement of greater than 60% of ASV.

At the start of the test, the total hardness of the blank control and 100 mg/L WAF test substance group were both 105 mg/L (calculated by the CaCO₃), and the salinity of the blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were all 0‰ during the test periods.

8.3 Hatching rate, surviving rate after post-hatched and cumulative abnormal rate of embryos / larvae

Results of hatching rate, surviving rate after post-hatched and cumulative abnormal rate are shown in Table 5.

The blastocyst stage of embryos (about 2 h-5.25 h) that not older than 8 h after fertilized were exposed. All embryos were completely hatched at 4^th day after start of the test, the hatching rate of embryos in blank control was 100%, which met the validity criterion that the hatching rate sould be more than 66%. The hatching rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 98%, 100%, 100%, 100% and 100%. The hatching rate of blank control had met the validity criterion that the hatched success should be more than 66%.

At the end of test, the surviving rate after post-hatched of blank control was 95%, which met the validity criterion that the surviving rate after post –hatched should be not less than 70%. The surviving rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 95%, 93%, 98%, 97% and 97%, respectively.

At the end of test, the cumulative abnormal rates of embryos / larvae or juvenile fish in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 5%,7%,3%, 2%, 3% and 3%.

8.4 The abnormal appearances or behaviours of embryos and larvae or juvenile fish

The results of abnormal appearances or behaviours are shown in Table 6-7.

During the test, no abnormal appearances or behaviours were observed in blank control and 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups.

8.5 The total lengths of surviving larvae or juvenile fish

The results of total lengths of surviving larvae or juvenile fish are shown in Table 8.

At the end of the test, the mean and the standard deviation of total length of surviving larvae or juvenile fish in blank control was 16.87mm±0.21 mm , coefficient of variation (CV) was 1.3%, which met the validity criterion that the CV should not exceed 20%.

The mean and standard deviation of total length of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 16.34 mm±0.40 mm, 16.38 mm±0.39 mm, 16.15 mm±0.54 mm, 16.43 mm±0.37 mm and 16.12 mm±0.50 mm, respectively.

8.6 The wet and dry weights of surviving larvae or juvenile fish

The results of wet weight(blotted dry) of surviving larvae or juvenile fish are shown in Table 9. At the end of the test, the average and standard deviation of wetweight of surviving larvae or juvenile fish in blank control was 47.9 mg±1.1 mg, the coefficient of variation was 2.2%, which was met the validity criterion that the coefficient of variation should not exceed 20%.

The means and standard deviation of wet weights in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 47.9 mg±4mg, 49.5 mg±2.6 mg, 44.8 mg±4 mg, 45.7 mg±1.4 mg and 45.5 mg±1.7 mg.

The results of dry weight of surviving larvae or juvenile fish are shown in Table 10. At the end of the test, the average and standard deviation of dry weight of surviving larvae or juvenile fish in blank control was 11.0 mg±0.39 mg, the coefficient of variation was 4%, which was met the validity criterion that the coefficient of variation should not exceed 20%.

The means and standard deviation of wet weights in 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were 11.3 mg±0.89 mg, 11.5 mg±0.57 mg, 10.6 mg±0.80 mg, 10.8 mg±0.69mg and 10.8 mg±0.54 mg.

8.7 The concentration of TOC in test solution

The limit of detection (LOD) and the limit of quantification (LOQ) of TOC analytical method were 0.2 mg/L and 0.8 mg/L, respectively. The linearity range of this test was 0.80 mg/L to 50.0 mg/L, the specific results are shown in Annex C.

The measured concentrations of TOC in blank control at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) ranged from 0.873 mg/L to 1.92 mg/L.

The measured concentrations of TOC in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) ranged from < 0.8 mg/L (LOQ) to 2.04 mg/L. The measured concentrations of TOC in all test substance groups were very similar to those of the blank control, indicating that test substance was poorly soluble in test water. See details in Table 11.

8.8 NOEL、LOEL and EL₁₀

The method of Chi-Square Test was used basing on the hatching rate, surviving rate after post-hatched and the abnormal rate, all the statistical results showed that the NOEL was 100 mg/L WAF, and the LOEL was greater than 100 mg/L WAF.

The total lengths, wet weights and dried weights were statistical, they were not according with the normal distribution or homogeneity of variance, so the non-parameter test of Jonckneere-Terpstra Test was used to statistical analysis of significant differences. Results showed that there statistically significant difference (P<0.05) based on total lengths between the blank control and 100 mg/L WAF test groups, therefore the NOEL was the loading rate of 32mg/L and the LOEL was the loading rate of 100 mg/L. However, there no statistical significant differences (p>0.05) between blank control and all test substance test groups based on the wet weights and dry weights, so the NOEL was the loading rate of 100 mg/L and the LOEL was greater than the loading rate of 100 mg/L.

The statistical method of linear regression model: Inhibition Concentration (ICp) Approach (Version 2.0) was used to calculate the value that causes x % inhibition of loading rate, like as ELx (e.g. EL₁₀, EL₂₀ or EL₃₀). In this test, the values of EL₁₀ were greater than loading rate of 100 mg/L based on the total lengths, wet weights and dry weights.

 

Validity criteria fulfilled:

yes

Conclusions:

The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (34 d), the NOEL, LOEL and EL10 of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:
Indicator EL10 Statistical method NOEL LOEL
Hatching rate — Chi-Square Test =100 mg/L >100 mg/L
Cumulative abnormal rate — Chi-Square Test =100 mg/L >100 mg/L
Surviving rate — Chi-Square Test =100 mg/L >100 mg/L
Total length of surviving fish >100 mg/L Jonckneere-Terpstra Test =32 mg/L =100 mg/L
Wet weight of surviving fish >100 mg/L Jonckneere-Terpstra Test =100 mg/L >100 mg/L
Dry weight of surviving fish >100 mg/L Jonckneere-Terpstra Test =100 mg/L >100 mg/L
Note：the Inhibition Concentration (ICp) Approach (Version 2.0) was used to calculate the value that causes x % inhibition of loading rate (EL10)

Executive summary:

Results

• Basis of the TOC as concentration analysis

LICOCARE RBW 300 FL TP with the character of low water solubility. And it’s not soluble in regular solvent (Methanol, acetonitrile, tetrahydrofuran, isopropanol, etc), so HPLC (High Performance Liquid Chromatography) and LC-MS/MS (Liquid Chromatography-Mass Spectrometer) is not suitable for analysis. According to the GC (Gas Chromatography) method in Certificate of Analysis that sponsor provided, the test substance should be derived first, but derivation must be proceeded under non-aqua system, which is not suitable for this test system and the analysis of water solution, meanwhile, the predicted limit of quantification (LOQ) of the analysis method would be high. Moreover, the carbon chain of some component of the test substance is much more than 40, so it’s not guaranteed that all constituents of test substance would be peaked in GC condition because of the high boiling point, and it would cause residual and pollute the apparatus. Consequently, in this test, only the concentrations of TOC in test solutions were measured to indicate the stability of exposed concentration of the test system.

(2) Basis of preparation of test solution

The LICOCARE RBW 300 FL TP with the character of Light yellow flakes solid, is a  complex mixture/UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in test water. The aqueous media was prepared following OECD guidance document No. 23 (2019), preparation the test solution by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF solution was separated by siphon. The WAF solution was checked for any undissolved or emulsified material by the Tyndall effect. Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). This procedure was followed for each renewal of the test solutions.

(3) The measured concentrations of TOC in test solutions

The limit of detection (LOD) and the limit of quantification (LOQ) of TOC were 0.2 mg/L and 0.8 mg/L, respectively. The linearity range of this test was 0.80 mg/L to 50.0 mg/L.

The measured concentrations of TOC in blank control at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) ranged from 0.873 mg/L to 1.92 mg/L.

The measured concentrations of TOC in nominal loading rates of 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L WAF test groups at 0 d, 7 d, 10 d, 14 d, 21 d, 27 d, 33 d (new solution) and 1 d, 8 d, 11 d, 15 d, 22 d, 28 d, 34 d (old solution) ranged from < 0.8 mg/L (LOQ) to 2.04 mg/L. The measured concentrations of TOC in all test substance groups were very similar to those of the blank control, indicating that test substance was poorly soluble in test water.

 (4) Toxicity effects on test organism

During the test periods, except the dead embryos or larvae, there no abnormal appearances or behaviours were observed. Under this test condition, at the end of the test (34 d), the No Observed Effect loading rate (NOEL) and Lowest Observed Effect loading rate (LOEL) and the 10% effective loading rate of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:

 

 

 

 

 

Indicator	EL10	NOEL	LOEL
Hatching rate	—	=100 mg/L	>100 mg/L
The abnormal rate of embryos/larvae	—	=100 mg/L	>100 mg/L
surviving rate	—	=100 mg/L	>100 mg/L
Total length of larvae/juvenile fish	>100 mg/L	=32 mg/L	=100 mg/L
The individual wet weight of larvae/juvenile fish (blotted-dry)	>100 mg/L	=100 mg/L	>100 mg/L
Dry weight of larvae/juvenile fish	>100 mg/L	=100 mg/L	>100 mg/L

Description of key information

The test substance is a kind of UVCB item, and insoluble in test water, so water accommodate fraction was used to prepare test solution and the test results were expressed as the nominal loading rate. Under this test condition, at the end of the test (34 d), the NOEL, LOEL and EL10 of Fish, Early-life Stage Toxicity Test to Rare Minnow (Gobiocypris rarus) are shown as follow:
Indicator EL10 Statistical method NOEL LOEL
Hatching rate — Chi-Square Test =100 mg/L >100 mg/L
Cumulative abnormal rate — Chi-Square Test =100 mg/L >100 mg/L
Surviving rate — Chi-Square Test =100 mg/L >100 mg/L
Total length of surviving fish >100 mg/L Jonckneere-Terpstra Test =32 mg/L =100 mg/L
Wet weight of surviving fish >100 mg/L Jonckneere-Terpstra Test =100 mg/L >100 mg/L
Dry weight of surviving fish >100 mg/L Jonckneere-Terpstra Test =100 mg/L >100 mg/L
Note：the Inhibition Concentration (ICp) Approach (Version 2.0) was used to calculate the value that causes x % inhibition of loading rate (EL10)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

total length of surviving fish

Effect concentration:

32 mg/L