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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2017 to 18 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Samples (approximately 150 mL) of the definitive test solutions and procedural controls were collected into Teflon® capped glass bottles (250 mL, no preservative). For the range-finding test, samples were collected immediately prior to test commencement (0 hour), after 48 hours and at test termination (96 hour) for analysis. For the definitive test, samples were collected immediately prior to test commencement (0 hour), after 48 hours (old and fresh solutions) and at test termination (96 hour) for analysis.
- All samples were refrigerated until transported to ISI and kept refrigerated by ISI until analysis was conducted. Samples collected at the beginning and end of the test were analysed within 24 hours of sample collection, whereas samples collected after 48 hours were analysed two days after sample collection, as these samples were collected on a Saturday and analysed the following Monday.
Vehicle:
no
Details on test solutions:
- Supporting documentation provided by the study sponsor, stated that the water solubility of the test material was 305 mg/L. However, preliminary testing involving analysis of the test material in dilution water (by ISI) gave a much lower limit of approximately 10 to 13 mg/L. This lower estimate was based on the analysis of a 1 g/L solution of the test material prepared in dilution water after 24 hours and 48 hours of continuous mixing. Based on these results, the highest tested nominal concentration of the test material in the range-finding and definitive tests was 1000 mg/L.
- The 1000 mg/L test solutions were prepared in 20 L glass aspirator bottles, stirred for approximately 24 hours at a rate sufficient to maintain a vortex between approximately 10 - 35% of the solution depth using a stir bar and stir plate. The solutions were then settled for approximately 1 hour. The first ~ 100 mL of solution removed from the glass aspirator was disposed of. All lower concentrations of the test material were prepared by further dilution of the 1000 mg/L test solution (i.e., addition of the appropriate amount of the 1000 mg/L solution with dilution water to achieve the desired nominal concentration). All test solutions were dispensed into 20-L plastic pails, each fitted with a Teflon liner.
- All tests included a procedural control (prepared in the same fashion as the test solution, except that no test material was added) and a negative control (i.e., 100% dilution water with no test material added).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Species: Oncorhynchus mykiss
- Age at study initiation: juvenile
- Length at study initiation: 3.43 ± 0.39 cm fork length (Range-finding); 3.62 ± 0.63 cm fork length (Definitive Test)

ACCLIMATION
- Acclimation period: The fish were acclimated to the laboratory conditions for at least twelve days prior to use in tests. 14-day acclimation took place in groundwater, there was < 5% mortality in 7 days prior to use in tests.
- Type and amount of food during acclimation: Commercial trout pellets. Feeding rate = 1 to 4% wet weight of fish

FEEDING DURING TEST
- No feeding took place during the test or in the preceding 24 hours before testing
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
200 mg/L as CaCO3
Test temperature:
15 ± 2°C
pH:
7.5 - 8.1
Dissolved oxygen:
9.0 - 9.8 mg/L
Nominal and measured concentrations:
Nominal: 62.5, 125, 250, 500 and 1000 mg/L
Measured: 0.85, 1.04, 2.47, 5.34, 9.89 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 20-L plastic pail with Teflon liner
- Fill volume: 15 L
- Aeration: 6.5 ± 1 mL/L/min. Dissolved oxygen > 60% of saturation in controls.
- Renewal rate of test solution: after 48 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The source of water used for holding and testing rainbow trout was moderately hard groundwater (water hardness ~350 mg/L as CaCO3, pH 7.8 - 8.1) from an aquifer in Aberfoyle, Ontario, Canada. Prior to use, the water was filtered through a 20-micron cellulose-acetate filter and diluted with Reverse Osmosis (RO) water to a hardness of 250 mg/L (as CaCO3). A continuous supply of oil-free compressed air was provided to bring the pH and concentrations of dissolved oxygen and other gases into equilibrium with air. The concentration of dissolved oxygen in the water was maintained at > 80% of the air saturation value. Water quality parameters (including pH, dissolved oxygen, conductivity and temperature) necessary for the survival of the test organisms were monitored and documented on a regular basis.
- Intervals of water quality measurement: Water quality parameters (including dissolved oxygen, pH, and conductivity) were measured daily in all test solutions. Dilution water hardness was measured at the beginning of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light, 8-hour dark (with 30 minute transition period)
- Light intensity: Ambient laboratory illumination, 100 to 500 lux

EFFECT PARAMETERS MEASURED: For all test solutions and controls, fish mortality was recorded after 24, 48, 72, and 96 hours of exposure. Test organisms were considered dead if there was no evidence of opercular or other activity and did not respond to subsequent gentle prodding.

RANGE-FINDING TEST
- A 1000 mg/L test solution was prepared by adding 20.0006 g of the test material into a 20 L glass aspirator bottle with 20 L of dilution water. The solution was stirred for approximately 24 hours and then settled for approximately 1 hour. Additional test solutions of 100 and 10 mg/L (nominal concentration) were prepared by adding 1.5 and 0.150 L of the 1000 mg/L into a 20 L graduated cylinder and making this up to a 15 L volume with dilution water.
- A total of 10 fish were exposed to each test and control treatments under static test conditions.

DEFINITIVE TEST
- The definitive test was initiated with renewal of the test solutions. Two 1000 mg/L test solutions were prepared by adding 20.0032 and 19.9986 g of the test material into each of two, 20 L glass aspirator bottles with 20 L of dilution water to achieve a sufficient volume of solution for testing purposes. The solutions were stirred for approximately 24 hours and then settled for approximately 1 hour. The solutions were then combined into a 50 L plastic tank with a new disposable plastic liner and mixed thoroughly prior to use in preparing the remaining test solutions. Additional nominal concentrations of the test material of 62.5, 125, 250, and 500 mg/L were prepared by adding 0.9375,1.875, 3.750 and 7.5 L of the 1000 mg/L into a 20 L graduated cylinder and each solution up to a 15 L volume with dilution water.
- Two additional 1000 mg/L test solutions were prepared (i.e., for use in the 48 hour renewal of test solutions) by adding 20.0026 and 20.0035 g of the test material into each of two, 20 L glass aspirator bottles with 20 L of dilution water to achieve a sufficient volume of solution for testing purposes. The additional test solutions were prepared as described for the t = 0 test solutions. The test solutions were dispensed into a set of duplicate 20 L plastic test vessels with Teflon liners.
- With the exception of the 1000 mg/L nominal concentration, transferring the test fish from the old solution after the initial 48 hours of exposure to the fresh solution was carried out by gently netting the fish from the old solution using an 8” dipping net, in the order of the lowest to the highest concentration (i.e., beginning with the controls). Due to the presence of dead fish in the 1000 mg/L concentration at 48 hours (n = 2 out of 10), the dead fish were first removed using an 8” dip net, then approximately 90% of the old test solution was decanted. The remaining live fish were then transferred to the fresh solution by gently pouring the remaining solution with the fish into an 8” dip net and transferring them to the fresh solution.
- A total of 10 fish were exposed to each test and control treatments.
Reference substance (positive control):
yes
Remarks:
Potassium chloride
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
660 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95 % C.L. = 578-753 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
707 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95 % C.L = 500-1000 mg/L
Details on results:
RANGE-FINDING TEST RESULTS
- Confirmation of Exposure Concentrations: Nominal, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning (0 h), middle (48 h) and end (96 h) of the test are provided in Table 1. Measured concentrations were below nominal concentrations due to the low solubility of the test material. Measured concentrations at the start of the test were generally within 20% of measured concentrations after 48 hours, but decreased below this by 96 hours. An unexplained increase in the measured concentration for the 10 mg/L nominal concentration was observed after 48 hours, but this decreased to below the < MDL of 0.1 mg/L by the end of the test.
- Mortality: The range-finding test met the test validity criteria. The percentage mortality of rainbow trout exposed to the test material during the range-finding test along with nominal concentrations are given in Table 2. 100% mortality of rainbow trout occurred within the first 72 hours exposure to a 1000 mg/L concentration of the test material, but no mortality (0%) was observed at all lower concentrations (≤ 100 mg/L). Additionally, there was no evidence of abnormal behaviour at these lower concentrations.

DEFINITIVE TEST RESULTS
- Confirmation of Exposure Concentrations: Measured concentrations of the test material at t = 0 hours were slightly higher than those of the corresponding solutions measured for the freshly prepared solutions at t = 48 hours. Additionally, the measured concentrations of the 48 hour old solutions were lower than the corresponding measured concentrations at the end of the test. These differences may be explained by the longer period of time between sample collection and analysis. The 48 hour old and 48 hour fresh solutions were collected on a Saturday and analysed following Monday, whereas the samples collected at the beginning and end of the test were analysed within 24 hours of sample collection. Overall these differences were considered to be reasonable given the low solubility of the test material.
- Mortality: The definitive test met the test validity criteria. A summary of the test results expressed in terms of the percent fish mortality observed in the test after 24, 48, 72 and 96 hours is summarised in Table 4. As can be seen in Table 4, 100% mortality of rainbow trout was observed at a nominal 1000 mg/L concentration of the test material, whereas little to no fish mortality (i.e., ≤ 10%) was observed at all lower concentrations during the 96 hour exposure. Additionally, there was no evidence of abnormal behaviour at these lower concentrations. The 24 and 48 hour LC50s were reported to be > 1000 mg/L. The 72 and 96 hour LC50s (and 95% confidence limits) were estimated to be 707 mg/L (500 - 1000) and 660 mg/L (578 - 753), respectively. The 96 hour LC0 and LC100 were 250 and 1000 mg/L, respectively.
Results with reference substance (positive control):
- Potassium chloride was the reference toxicant used in this study. The LC50 was compared to historical data and was deemed acceptable based on results falling within the 95 % confidence interval (warning limit) calculated for the last twenty (20) reference tests.
- The 96-hour LC50 for the rainbow trout reference toxicant test associated with the definitive test was 3.737 g/L.
Reported statistics and error estimates:
- Nominal concentrations were used to determine the test endpoints. Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance.
- Estimates of the 24, 48, 72 and 96-hour LC50s from the results of the range-finding test were estimated from a visual inspection of the data. Estimates of the 24 and 48- hour LC50s from the definitive test were estimated by visual inspection of the data. Estimates of the 72 and 96-hour LC50s were estimated by the Binomial and Spearman-Karber, respectively (CETIS, 2013).
Sublethal observations / clinical signs:

Table 1: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material for the Range-Finding Test

Nominal Concentration

(mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

48 Hours

96 Hours

1000

10.38c

8.58d

7.78e

8.81

100

1.02

0.97

0.7

0.91

10

0.12

0.19

< MDL

0.15

0 (Procedural Control)

< MDL

< MDL

< MDL

< MDL

c Average of duplicate analyses. The Relative Percent Difference (RPD) = 0.6%

d Average of duplicate analyses. RPD = 4.6%

e Average of duplicate analyses. RPD = 3.0%

Method Detection Limit (MDL) = 0.1 mg/L

 

Table 2: Mortality of Rainbow Trout After 24, 48, 72 and 96 Hours Exposure to the Test Material During the Range-Finding Test

Nominal Concentration

(mg/L)

Percent Mortality

24 Hours

48 Hours

72 Hours

96 Hours

1000

0

60

100

100

100

0

0

0

0

10

0

0

0

0

0 (Procedural Control)

0

0

0

0

0 (Negative Control)

0

0

0

0

 

Table 3: Summary of the Nominal, Measured and Time-Weighted Mean Concentrations of the Test Material During the Definitive Test

Nominal Concentration

(mg/L)

Measured Concentration (mg/L)

Time-Weighted Mean Concentration (mg/L)

0 Hours

48 Hour Old

48 Hour Fresh

96 Hours

1000

13.25d

7.8e

9.76f

9.23g

9.89

500

8.29

2.87

6.45

4.76

5.34

250

3.86

1.8

2.51

1.98

2.47

125

1.64

0.77

0.93

0.91

1.04

62.5

0.85

N/A

N/A

N/A

0.85i

0 (Procedural Control)

< MDL

< MDL

< MDL

< MDL

< MDL

0 (Negative Control)

< MDL

< MDL

< MDL

< MDL

< MDL

d Result of duplicate analysis. Relative Percent Difference (RPD) 2.3%

e Result of duplicate analysis. RPD = 7.1%

f Result of duplicate analysis. RPD = 3.3%

g Result of duplicate analysis. RPD = 4.2%

i Based on a single measurement (i.e., not a TWM)

Method Detection Limit (MDL) = 0.1 mg/L

 

Table 4: Mortality of Rainbow Trout After 24, 48, 72 and 96 Hours Exposure to the Test Material During the Definitive Test

Nominal Concentration

(mg/L)

Percent Mortality

24 Hours

48 Hours

72 Hours

96 Hours

1000

0

20

100

100

500

0

0

0

0

250

0

0

0

0

125

0

0

0

0

62.5

0

0

0

0

0 (Procedural Control)

0

0

0

0

0 (Negative Control)

0

0

0

0

 

TEST VALIDITY CRITERIA

The range-finding and definitive tests met the test validity criteria:

- Mortality and impairment of the control fish ≤ 10% at the end of the test.

- Constant conditions were maintained throughout the test.

- Dissolved oxygen concentration were ≥ 60% of the air saturation value at the temperature of 15 ± 1°C throughout the test.

- Based on the low water solubility and the UVCB nature of the test material, from the measured concentration data, it is clear that only some of the constituents of the test material dissolved in the test solutions. This is not atypical with UVCBs of low water solubility. Due to some instability observed, static renewal of test solutions at 48 hours was deployed in the definitive tests. Based on a review of the nominal and TWM concentrations, the test material was generally maintained throughout the test.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, 24 and 48 hour LC50s were reported to be > 1000 mg/L, as there was little to no fish mortality (i.e., ≤ 20%) at any concentration during the first 48 hours of the test. The 72 and 96 hour LC50s (and 95% confidence limits) were estimated to be 707 mg/L (500 - 1000) and 660 mg/L (578 - 753), respectively. The 96 hour LC0 and LC100 were 250 and 1000 mg/L, respectively.
Executive summary:

The acute toxicity of the test material to fish was investigated in accordance with the standardised guidelines OECD 203, under GLP conditions.

Both range-finding and definitive tests met the validity criteria.

Analytical concentrations were verified in this study, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

Results of the range-finding test indicated that the 96-hour LC50 for the test material was in the range of 100 to 1000 mg/L. Additionally, the test showed that the measured concentration of the test material decreased less than 20% after 48 hours, but more than 20% after 48 hours. As such, the definitive test was conducted as a multiple concentration test with renewal of the test solutions after 48 hours.

Results of the definitive test showed that survival of rainbow trout was not adversely affected at nominal test material concentrations below 500 mg/L.

Under the conditions of this study, 24 and 48 hour LC50s were reported to be > 1000 mg/L, as there was little to no fish mortality (i.e., ≤ 20%) at any concentration during the first 48 hours of the test. The 72 and 96 hour LC50s (and 95% confidence limits) were estimated to be 707 mg/L (500 - 1000) and 660 mg/L (578 - 753), respectively. The 96 hour LC0 and LC100 were 250 and 1000 mg/L, respectively.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 August 2018 to 24 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 203 Fish Acute Toxicity Test [M].
Version / remarks:
Second Edition Beijing: China Environmental Press. 2013, page 30-36.
The editorial board of “The Guidelines for the Testing of Chemicals” of Chemical Registration Center of the Ministry of Environmental Protection.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Standardization Technical Committee for the Management of Hazardous Chemicals. GB/T 27861-2011 Chemicals Fish Acute Toxicity Test [S].
Version / remarks:
Beijing: China Standard Press. 2012.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Standardization Technical Committee for the Management of Hazardous Chemicals. GB/T 29763-2013 Chemicals Rare Minnow (Gobiocypris rarus) Acute Toxicity Test [S].
Version / remarks:
Beijing: China Standard Press. 2013.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Department of Science and Technology Standards of State Environmental Protection Administration. HJ/T153-2004 Guidelines for Testing of Chemicals [S].
Version / remarks:
Beijing: China Standard Press. 2004.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Samples were taken for analysis at the beginning of exposure (0 h), at the end of exposure (96 h) and at 24, 48, 72 hours before and after test solution renewal.
- On each occasion, four samples (one sample was retained by cold storage in case re-analysis was required) were taken from test material treatment group and one from blank control group for LC/MS/MS analysis. Three samples (one sample was retained in case reanalysis was required) were taken from test material treatment group and control group for TOC analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- The test solution was prepared by WAF method. The test material was accurately weighed and added to 10 L test water to give the loading rate of 100 mg/L. After the addition of test material, the solution was stirred by magnetic stirrer using a stirring rate at approximately 15 % vortex height of the liquid depth in the test vessel. The stirring was maintained for 24 hours, followed by settling for about 24 hours. The siphon sealed by a piece of Parafilm was submerged into the solution avoiding insoluble test material on the surface. And then, an iron wire was passed through the siphon from inside and pushed off the parafilm seal. About 7 L of solution was withdrawn from the middle layer of solution (the first 100 mL was discarded) and used for exposure. The test solution was freshly prepared before use. Microscopic observation was performed before use and insoluble small droplets were not observed.
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
TEST ORGANISM
- Common name: rare minnow (Gobiocypris rarus)
- Characteristics which make this test organism suitable for toxicity testing are its ease of obtaining, raising, sensitivity to a variety of chemical substances and the extensive data base that exists for this species.
- Source: Rare minnow were reproduced and maintained in the water renewal system of CTI-SET
- Age at study initiation: fish were approximately the same age
- Length: 2.87 – 3.24 cm (mean ± SD: 3.07 ± 0.14 cm)
- Weight: 300 – 335 mg (mean ± SD: 319 ± 12 mg)
- The fish were healthy and free from any apparent malformation.

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions: The fish were acclimated in the water having the same quality as the test water. During the seven days acclimation before the test, the water temperature was maintained in the range of 21-25 °C and the change was not more than 2 °C; 12 hours light was given daily; dissolved oxygen concentration was above 80 % of air saturation value (ASV).
- The fish were fed daily until 24 hours before the test.
- During the 7 days acclimation prior to the test, the total mortality was 0 %. The fish could be used.

FEEDING DURING TEST: No
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
150 mg/L (as CaCO3)
Test temperature:
24.0 - 24.7 °C
pH:
7.61 - 8.36
Dissolved oxygen:
71.4 - 97.4 % ASV
Nominal and measured concentrations:
Nominal: loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 7 L
- Renewal rate of test solution: the test solution was renewed every 24 hours during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: < 1g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The tap water dechlorinated by aeration (also named as treated water) was used as test water, pH value of the water was in the range of 6.0-8.5 and the total hardness was in the range of 10-250 mg/L (calculated as CaCO3).
- Intervals of water quality measurement: The temperature, pH and concentration of dissolved oxygen of the test solution in control and test material treatment groups were measured every 24 hours. The total hardness of the test water was measured at the start of the study.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours photoperiod daily

EFFECT PARAMETERS MEASURED
- At the end of the test, the fish in the blank control group were used to measure the body length (total length) and wet-weight.
- Fish were inspected for mortalities or visible abnormalities after 3, 24, 48, 72 and 96 hours. Criteria of death were absence of respiratory movement and lack of response to external stimulation of the caudal peduncle. The dead fish were immediately removed, stored temporarily in the refrigerator and then delivered to the approved waste handling company for disposal.

TEST CONCENTRATIONS
- Preliminary test: The test material had no toxic effect on the fish at nominal loading rate of 100 mg/L. The limit test could be conducted at the nominal loading rate of 100 mg/L in definitive test.
- Based on the result of the preliminary test, the limit test was performed at the loading rate of 100 mg/L, and blank control was set in parallel.
Reference substance (positive control):
yes
Remarks:
potassium bichromate
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
mortality (fish)
Details on results:
OBSERVATIONS
- During the period of exposure, there was no mortality and abnormal symptom in the blank control and loading rate of 100 mg/L.

CONCENTRATION ANALYSIS
- Result of concentration analysis by LC/MS/MS: The test material was not detected in the blank control group. The concentration of test material in the old solutions was not maintained in the range of 80-120 % of that in the new solutions. The geometric mean of measured concentration was 1.3 mg/L for the loading rate of 100 mg/L.
- TOC concentration: The TOC concentration of test material corrected by the blank control was in the range of 2.20-3.01 mg/L.
- As the test material is UVCB in nature, the dissolved portion may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

LL50
- In this study, based on the loading rate, the 96h-LL50 of test material to rare minnow was > 100 mg/L.

VALIDITY OF THE TEST
- At the end of the test, the mortality in the control was 0 %, not exceeding 10 %. During the test, the concentration of dissolved oxygen was in the range of 71.4-97.4 %, not falling below 60 % of the air saturation value. Temperature and pH were maintained quite constant during the exposure period.
- Measured concentrations of the test material were not maintained in the 80 - 120 % range. However, as the test material is UVCB in nature, the dissolved portion may have been one or several of its components. For this reason, results are based on nominal loading rates only.
Results with reference substance (positive control):
- The result of the latest sensitivity test (Study No.:18ST003) conducted from 16 April 2018 to 20 April 2018 indicated that the 96h-LC50 of potassium bichromate (CAS No.: 7778-50-9) to Rare minnow was 205 mg/L and its 95 % confidential limit was 184-228 mg/L, which was within the range of 135.73-416.93 mg/L based on the result of lab ring test.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 96 h-LL50 of test material to rare minnow was more than 100 mg/L, based on WAF loading rate.
Executive summary:

The short-term toxicity of the test material to fish was investigated in accordance with the standardised guideline OECD 203 and other Chinese guidelines, under GLP conditions.

The purpose of this study was to estimate the acute toxicity of the test material to rare minnow (Gobiocypris rarus). According to the result of preliminary test, the limit test was performed using semi-static exposure procedure. Due to the low water solubility of the test material, a WAF (water accommodated fraction) approach was used in testing. The WAF loading rate of test material was 100 mg/L and a blank control was set in parallel. Seven fish were used for each group and there was no replicate. The fish were observed at 3, 24, 48, 72 and 96 hours of exposure. The mortality and abnormal behaviour were recorded. According to the mortality of test fish, the LL50 was estimated.

The concentration of test material in test solutions was analysed by LC/MS/MS and TOC analyser at the beginning of exposure (0 hour), at the end of exposure (96 hours) and at 24, 48, 72 hours before and after test solution renewal.

During the period of exposure, there was no mortality and abnormal symptom in the blank and test material treatment group.

Analysis by LC/MS/MS showed that the test material was not detected in the blank control group. The concentration of test material in the old solutions was not maintained in the range of 80-120 % of that in the new solutions. The geometric mean of measured concentration was 1.3 mg/L for the loading rate of 100 mg/L. The TOC concentration of test material corrected by the blank control was in the range of 2.20-3.01 mg/L. As the test material is UVCB in nature, the dissolved portion may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Under the conditions of this study, the 96 h-LL50 of test material to rare minnow was more than 100 mg/L, based on WAF loading rate.

Description of key information

Holtze (2018)

Under the conditions of this study, the 96 -h LL50 of the test material to rainbow trout was 660 mg/L, based on WAF loading rate.

Baolian (2018)

Under the conditions of this study, the 96 h-LL50 of test material to rare minnow was more than 100 mg/L, based on WAF loading rate.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

Baolian (2018)

The short-term toxicity of the test material to fish was investigated in accordance with the standardised guideline OECD 203 and other Chinese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The purpose of this study was to estimate the acute toxicity of the test material to rare minnow (Gobiocypris rarus). According to the result of preliminary test, the limit test was performed using semi-static exposure procedure. Due to the low water solubility of the test material, a WAF (water accommodated fraction) approach was used in testing. The WAF loading rate of test material was 100 mg/L and a blank control was set in parallel. Seven fish were used for each group and there was no replicate. The fish were observed at 3, 24, 48, 72 and 96 hours of exposure. The mortality and abnormal behaviour were recorded. According to the mortality of test fish, the LL50 was estimated.

The concentration of test material in test solutions was analysed by LC/MS/MS and TOC analyser at the beginning of exposure (0 hour), at the end of exposure (96 hours) and at 24, 48, 72 hours before and after test solution renewal.

During the period of exposure, there was no mortality and abnormal symptom in the blank and test material treatment group.

Analysis by LC/MS/MS showed that the test material was not detected in the blank control group. The concentration of test material in the old solutions was not maintained in the range of 80-120 % of that in the new solutions. The geometric mean of measured concentration was 1.3 mg/L for the loading rate of 100 mg/L. The TOC concentration of test material corrected by the blank control was in the range of 2.20-3.01 mg/L. As the test material is UVCB in nature, the dissolved portion may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Under the conditions of this study, the 96 h-LL50 of test material to rare minnow was more than 100 mg/L, based on WAF loading rate.

Holtze (2018)

The acute toxicity of the test material to fish was investigated in accordance with the standardised guidelines OECD 203, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Both range-finding and definitive tests met the validity criteria.

Analytical concentrations were verified in this study, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only.

Results of the range-finding test indicated that the 96-hour LC50 for the test material was in the range of 100 to 1000 mg/L. Additionally, the test showed that the measured concentration of the test material decreased less than 20% after 48 hours, but more than 20% after 48 hours. As such, the definitive test was conducted as a multiple concentration test with renewal of the test solutions after 48 hours.

Results of the definitive test showed that survival of rainbow trout was not adversely affected at nominal test material concentrations below 500 mg/L.

Under the conditions of this study, 24 and 48 hour LC50s were reported to be > 1000 mg/L, as there was little to no fish mortality (i.e., ≤ 20%) at any concentration during the first 48 hours of the test. The 72 and 96 hour LC50s (and 95% confidence limits) were estimated to be 707 mg/L (500 - 1000) and 660 mg/L (578 - 753), respectively. The 96 hour LC0 and LC100 were 250 and 1000 mg/L, respectively.