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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:
06 April 2014 to 24 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
RANGE-FINDING TEST
- A sample of each test concentration was taken for chemical analysis at 0 and 24 hours.
- All samples were stored frozen prior to analysis.

CHEMICAL ANALYSIS OF TEST CONCENTRATIONS IN THE DEFINITIVE TEST
- Water samples were taken from the control and all surviving test groups for quantitative analysis at 0 and 72 hours (fresh media) plus 72 and 96 hours (old media). The samples were stored frozen prior to analysis.
- Duplicate samples taken at 0 and 72 hours (fresh media) plus 72 and 96 hours (old media) were stored frozen for further analysis if necesary.
- Samples were also taken at 24 hours (fresh media), 48 hours (old and fresh media) and 72 hours (old media). These samples were stored frozen for further analysis if necesary.
- Only samples at the No Observed Effect Concentration and above were analysed.

TOTAL ORGANIC CARBON ANALYSIS IN THE DEFINITIVE TEST
- Water samples were taken from the control and each surviving test group at 0 and 72 hours (fresh media) plus 24 and 96 hours (old media).
- Duplicate samples taken at 0 and 72 hours (fresh media) plus 72 and 96 hours (old media) were stored at approximately 4 °C for further analysis if necesary.
- Samples were also taken at 24 hours (fresh media), 48 hours (old and fresh media) and 72 hours (old media). These samples were stored at approximately 4 °C for further analysis if necesary.
Details on test solutions:
TEST WATER
- The test water used for range-finding and definitive tests was the same as that used to maintain stock fish.
- Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener).
- After dichlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Typical water quality characteristics for the tap water as supplied, prior to dichlorination and softening, are given in Appendix 1 (attached).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss).
- Fish were obtained from Brow Well Fisheries Limited, Hebden near Skipton, Yorkshire, UK,
- Fish were maintained in-house from 30 September 2014 in a glass fibre tank with a ‘single pass’ water renewal system.
- The fish were acclimatised to test conditions from 03 October 2014 to 13 October 2014.
- The lighting cycle was controlled to give 16 hours light and 8 hour darkness with 20 minute dawn and dusk transition periods.
- Water temperature was controlled at approximately 14 °C with a dissolved oxygen content ≥ 8.9 mg O2/L. These parameters were recorded daily.
- Stock fish were fed commercial trout pellets and feeding was discontinued approximately 24 hours prior to the start of the definitive test.
- No mortality took place in the 7 days prior to the start of the test.
- Fish had a mean standard length of 4.3 cm (sd = 0.3) and a mean weight of 1.07 g (sd = 0.31) at the end of the definitive test. Based on the mean weight, the loading rate was 0.37 g bw/L.
- Diet and diluent water were considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
- approximately 140 mg/L as CaCO3
Test temperature:
14 °C
pH:
7.6 to 8.7 (see Table 5, attached)
Dissolved oxygen:
9.6 to 9.9 mg O2/L (see Table 5, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal test item concentrations of 1.0, 10 and 100 mg/L.

DEFINITIVE TEST
- Test item concentration of 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution.
Details on test conditions:
VALIDATION OF MIXING PERIOD
- Preliminary solubility work indicated that the test item was practically insoluble in water using traditional methods of preparation such as ultrasonication and high shear mixing.
- Based on this information, the test substance was categorised as a ‘difficult substance’ in line with the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD; 2000).
- A media preparation trial was therefore conducted to determine the solubility of the test item under test conditions (see Appendix 2, attached).

RANGE-FINDING TEST
- test item concentrations to be used in the definitive study were determined by a preliminary range-finding test.
- Fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
- Nominal amounts of test item (22, 220 and 2200 mg) were each separately added to the surface of 22 L of test water to give 1.0, 10 and 100 mg/L loading rates.
- After addition of test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one hour.
- A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was then inserted into the glass tube and pushed through the Nescofilm seal.
- The aqueous phase was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rates.
- Microscopic inspection of the aqueous phase showed no micro-dispersions or undissolved test item to be present.
- Three fish were added to each 20 L test and control vessel and maintained for 96 hours under static test conditions. The test was conducted at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness and 20 minute dawn/dusk transition periods.
- The control group was maintained under identical conditions but not exposed to the test material.
- Mortality and sub-lethal effects of exposure were determined by visual inspection of fish after 3, 6, 24, 48, 72 and 96 hours.

DEFINITIVE TEST
- A nominal amount of test item (1100 mg) was dispensed onto the surface of 11 L of test water prior to stirring for 23 hours with the aid of magnetic stirring at a rate such that a dimple was formed at the water surface.
- After stirring the solution was allowed to stand for one hour.
- A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was then inserted into the glass tube and pushed through the Nescofilm seal.
- The aqueous phase was removed by mid-depth siphoning (the first 75-100 mL discarded) to give a 100 % v/v saturated solution.
- Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 and 72 hours (fresh media) plus 24 and 96 hours (old media). Details of the analysis are given in Appendix 3 (attached).
- The concentration of boron in the test preparations were verified by chemical analysis at 0 and 72 hours (fresh media) plus 24 and 96 hours (old media). Details of the analysis are given in Appendix 4 (attached).
- A series of dilutions was made from the saturated solution to give the required test concentrations of 10, 5.6, 3.2, 1.8 and 1.0 % v/v saturated solution.
- Glass exposure vessels (25-30 L) were used for each test concentration.
- At the start of the test, seven fish were placed into the test preparations in each vessel at random.
- Test vessels were then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours dark and 20 minute dark/dusk transition periods.
- The test vessels were aerated via narrow bore glass tubes.
- Fish were not individually identified and received no food during exposure to the test item.
- The control group was maintained under identical conditions but not exposed to the test material.
- A semi-static regime was employed in the test involving daily renewal of test preparations to prevent build-up of nitrogenous waste products.

TEST ORANISM OBSERVATIONS
- Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure.
- The criteria for death were absence of both respiratory movement and response to physical stimulation.

WATER QUALITY CRITERIA
- Water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.
- Measurements at 0 hours and after each test media renewal at 24, 48 and 72 hours represented those of fresh test preparations while measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represented the used (24-hour old) test preparations.
- Dissolved oxygen concentration and pH were measured using a Hach HQ30d Flexi handheld meter whilst temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of each mixing period.

VALIDATION CRITERIA
- In the control, not more than one of the fish should die or show signs of stress during the 96 hours.
- Dissolved oxygen concentration at the end of the test should be ≥ 60 % of air saturation values (ASV) in the control and test vessels.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: carbon content of test material
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits 1.1 to 1.6 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: carbon content of test material
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: carbon content of test material
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
8.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: boron content of test material
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits 6.4 to 11 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
6.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: boron content of test material
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: boron content of test material
Basis for effect:
mortality
Details on results:
RANGE-FINDING TEST
- Cumulative mortality data from exposure of rainbow trout to the test item are given in Table 1 (attached) and sub-lethal effects are given in Table 2 (attached).
- No mortalities were seen at the 1.0 mg/L loading rate. However, mortalities were observed at the 10 and 100 mg/L loading rates.
- After approximately 3 minutes exposure, all of the fish at the 100 mg/L loading rate were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1996 Amendment Regulations 2012) the fish were killed and classed as mortalities for the following observational time point.
- After approximately 30 minutes exposure, all of the fish at the 10 mg/L loading rate were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1996 Amendment Regulations 2012) the fish were killed and classed as mortalities for the following observational time point.
- Based on this information, test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution were selected for the definitive test.
- Chemical analysis of the test preparations at 0 and 24 hours showed measured boron concentrations of between 0.022 and 2.2 mg/L (see Appendix 4, attached).

DEFINITIVE TEST
- Chemical analysis of the 5.6 and 10 % v/v saturated solution test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured boron concentrations ranging from 0.13 to 0.24 mg/L.
- The measured boron concentrations were equivalent to test item concentrations of 6.06 to 11.08 mg/L based on a test item boron content of 2.146 % (see Appendix 4, attached).
- Given that the boron concentration of the test item remained stable over the test duration, it was considered appropriate to base the results on the 0-hour equivalent test item concentrations.
- Total Organic Carbon (TOC) analysis of the 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured carbon concentrations of less than the limit of quantification of the analytical method (LOQ 1.0 mg C/L to 3.01 mg C/L; equivalent to test item concentrations of less than LOQ to 4.0 mg/L based on test item carbon content of 75.78 %).

MORTALITY DATA
- Cumulative mortality data from exposure of rainbow trout to the test item during the definitive test are given in Table 3 (attached).
- The relationship between percentage mortality and nominal concentration at 96 hours is given in Figure 1 (attached).
- Inspection of mortality data at 3, 6 and 24 hours and analysis of the mortality data by the geometric mean method at 48, 72 and 96 hours gave the results shown below based on carbon content and the time weighted mean / geometric mean measured test concentrations.
- Results of the definitive test based on carbon content showed the highest test concentration resulting in 0 % mortality was 1.1 mg/L and the lowest test concentration resulting in 100 % mortality was 1.6 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 1.6 mg/L carbon and the No Observed Effect Concentration (NOEC) was 1.1 mg/L carbon.
- The relationship between the median lethal concentration (LC50) and time is presented graphically in Figure 2 (attached).
- Inspection of mortality data at 3, 6 and 24 hours and analysis of the mortality data by the geometric mean method at 48, 72 and 96 hours gave the results shown below based on boron content and the 0-hour equivalent test concentrations.
- Results of the definitive test showed the highest test concentration based on boron content showed the highest test concentration resulting in 0 % mortality was 6.4 mg/L and the lowest test concentration resulting in 100 % mortality was 11 mg/L. The Lowest Observed Effect Concentration (LOEC) was considered to be 11 mg/L boron and the No Observed Effect Concentration (NOEC) was 6.4 mg/L boron.
- The relationship between the median lethal concentration (LC50) and time is presented graphically in Figure 3 (attached).

SUB-LETHAL EFFECTS
- Sub-lethal effects of exposure were observed at the 10 % v/v saturated solution test concentration. These responses were swimming at the bottom of the vessel, a slight loss of equilibrium, swimming at the surface and increased pigmentation (see Table 4, attached).
- After approximately 48 hours exposure all fish at 10 % v/v saturated solution were observed to exhibit prolonged sub-lethal effects. Due to animal welfare implications (Animals (Scientific Procedures) Act 1996 Amendment Regulations 2012) the fish were killed and classed as mortalities for the following observational time point.

VALIDATION CRITERIA
- The test was considered to be valid given that none of the control fish died or showed signs of stress during the test.
- The test was met the validity criterion for oxygen concentration, which was ≥ 60 % of ASV (equivalent to 6.2 mg O2/L) in the control and test vessels.

WATER QUALITY CRITERIA
- The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during mixing and testing of the saturated solution.
- At the start of the mixing period the saturated solution was observed to be a clear colourless water column with oily globules of test item at the surface.
- After 23 hours stirring and a one hour standing period, the 100 % v/v saturated solution was observed to remain as at the start of stirring.
- Microscopic inspection of the aqueous phase showed no micro-dispersions or undissolved test item to be present.
- After siphoning, and for the duration of the test, the 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution test concentrations were observed to be clear, colourless, solutions.
Reported statistics and error estimates:
- The LC50 value and associated confidence limits at 48, 72 and 96 hours were calculated using the geometric mean method.
- The LC50 value = square root of C1 x C2 where C1 = concentration showing 0 % mortality and C2 = concentration showing 100 % mortality.
- If there were no concentrations showing mortalities between 0 % and 100 %, the geometric mean of the highest concentration showing no lethality and the lowest concentrations showing 100 % was calculated.
- The concentrations resulting in 0 % and 100 % mortality gave the 95 % confidence limits.

RESULTS BASED ON CARBON CONTENT

Time (h)

LC50 (mg/L)

95 % confidence limits

3

> 1.6

-

6

> 1.6

-

24

> 1.6

-

48

1.3

1.1 to 1.6*

72

1.3

1.1 to 1.6*

96

1.3

1.1 to 1.6*

* Test concentrations resulting in 0 % and 100 % mortality

RESULTS BASED ON BORON CONTENT

Time (h)

LC50 (mg/L)

95 % confidence limits (mg/L)

3

> 11

-

6

> 11

-

24

> 11

-

48

8.4

6.4 to 11*

72

8.4

6.4 to 11*

96

8.4

6.4 to 11*

* Test concentrations resulting in 0 % and 100 % mortality

Validity criteria fulfilled:
yes
Conclusions:
Exposure of rainbow trout to the test item gave an LC50 (96 h) value of 1.3 mg/L (95 % confidence limits 1.1 to 1.6 mg/L) based on carbon content. The No Observed Effect Concentration (NOEC) based on carbon content was 1.1 mg/L and the Lowest Observed Effect Concentration (LOEC) based on carbon content was 1.6 mg/L. With respect to boron content, the LC50 (96 h) value was determined to be 8.4 mg/L (95 % confidence limits 6.4 to 11 mg/L). The No Observed Effect Concentration (NOEC) based on boron content was 6.4 mg/L and the Lowest Observed Effect Concentration (LOEC) based on boron content was 11 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203 “Fish, Acute Toxicity Test” referenced as Method C.1 of Commission Regulation (EC) No 440/2008.

 

METHODS

Due to the low aqueous solubility and pure nature of the test item, the test medium was prepared as a slow stir saturated solution for the purposes of the definitive test. Following a preliminary range-finding test, fish were exposed, in groups of seven, to the test item over a range of nominal concentrations (1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution) for a period of 96 hours at a temperature of 14 °C under semi-static conditions. The number of mortalities and any sub-lethal effects of exposure were determined in each test and control vessel at 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

RESULTS

Chemical analysis of the 5.6 and 10 % v/v saturated solution test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured boron concentrations of 0.13 to 0.24 mg/L (equivalent to test item concentration of 6.06 to 11.09 mg/L based on a test item boron content of 2.146 %). Total Organic Carbon (TOC) analysis of the 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured carbon concentrations of less than the limit of quantification of the analytical method (LOQ 1.0 mg C/L to 3.01 mg C/L; equivalent to test item concentrations of less than LOQ to 4.0 mg/L based on test item carbon content of 75.78 %).

 

CONCLUSION

Exposure of rainbow trout to the test item gave an LC50 (96 h) value of 1.3 mg/L (95 % confidence limits 1.1 to 1.6 mg/L) based on carbon content. The No Observed Effect Concentration (NOEC) based on carbon content was 1.1 mg/L and the Lowest Observed Effect Concentration (LOEC) based on carbon content was 1.6 mg/L. With respect to boron content, the LC50 (96 h) value was determined to be 8.4 mg/L (95 % confidence limits 6.4 to 11 mg/L). The No Observed Effect Concentration (NOEC) based on boron content was 6.4 mg/L and the Lowest Observed Effect Concentration (LOEC) based on boron content was 11 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07 January 2015 to 03 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Systems [M]. 2nd edition. Beijing: China Environment Press. 2013: 30-36.
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 27861-2011, Chemicals-Fish acute toxicity test, Beijing: SAC, 2011.
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 29763-2013, Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test Beijing: SAC, 2013.
Deviations:
no
Qualifier:
according to
Guideline:
other: Ecological Effects Test Guidelines, OPPTS 850.1075. Fish Acute Toxicity Test, Freshwater and Marine(S]. EPA 712-C-96-118, 1996.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Water samples (20.0 mL) were taken (at least in duplicate) from each concentration during the definitive test at 0, 24, 48, 72 and 96 hours.
- On each occasion, one sample was analysed after certain pre-treatments and remaining samples were retained to permit further analysis if necessary.
Vehicle:
no
Details on test solutions:
DILUTION MEDIUM
- Good quality tap water, which had been dechlorinated for at least 24 hours, was used in the test.
- Total hardness of the water was 145.0 mg/L as CaCO3.
- Oxygen concentration of the water was approximately 98 % of the air saturation value and pH was 7.82 at room temperature.
- Characteristics of the dilution water were measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The most recent measuring result is shown in Table 9 (attached).
Test organisms (species):
other: Globiocypris rarus
Details on test organisms:
- The test species, Globiocypris rarus (Batch F20141215) was obtained from a fish supplier of the Institute of Hydrobiology, Chinese Academy of Sciences.
- Fish were kept in holding tanks supplied with a continuous flow of aerated water for at least 12 days before being used for testing.
- Fish had been held for 23 days before the start of the range-finding test and 46 days before the start of the definitive test.
- Fish were held in water of the quality and temperature to be used in the test for 7 days prior to the investigation.
- A photoperiod of 16 hours light (provided by fluorescent tubes) and 8 hours dark was maintained.
- Oxygen concentration was 80 % of the air saturation value.
- Fish were fed on proprietary fish food daily during the holding period. They were then held without food for approximately 24 hours before being placed into the test vessels.
- The ingredients of the fish food were crude protein (> 36 %); crude fat (> 2.0 %); crude fibre (< 3.0 %); crude ash (< 12.0 %); moisture (< 10.0 %).
- Characteristics of the fish food were measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The most recent results are shown in Table 10 (attached).
- Tanks were inspected daily during the holding period and any debris or unhealthy/dead fish were removed.
- No mortality was observed in the 7 days after a 48-hour acclimation period and the batch of fish was accepted.
- The average wet weight of the fish used in the test was 0.286 g (standard deviation 6.94 %) and the average length of the fish was 2.59 cm (7.34 %) (see Table 1, attached).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
136 to 145 mg/L as CaCO3 (see Table 4, attached)
Test temperature:
23.1 to 23.5 °C during the definitive test (see Table 4, attached)
pH:
7.91 to 8.23 during the definitive test (see Table 4, attached)
Dissolved oxygen:
73 % to 93 % during the definitive test (see Table 4, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal concentrations of 1.0, 10.0 and 100 mg/L

DEFINITIVE TEST
- Nominal concentrations of 10 %, 20 %, 40 %, 60 %, 80 % and 100 % v/v saturated solution
Details on test conditions:
APPARATUS
- Oxygen meter, thermometer and pH meter (HACH HQ40d)
- Hardness of water (HACH Model 16900).
- Analytic balance (METTLER TOLEDO AG135, Switzerland, accuracy 0.1 mg)
- Tanks made of chemically inert material with a sealable inert lid and with a capacity of approximately 5 L (Haimen Sanhe Zuping Glass Instrument Factory, Jiangsu).
- Thermostatic water bath (Chang Yuan Medical Instrument Factory, Jiangsu).
- ICP-MS (Thermo Scientific X Series 2, USA).

REFERENCE SUBSTANCE
- Substance: Potassium dichromate (K2CrO7)
- CAS number: 7778-50-9
- Purity: ≥ 99.8 %
- Lot number: NH20131115
- Supplier: Nanjing Chemical Reagent Co Ltd
- The test with the reference substance was performed at least once for each batch of fish as a means of assuring the laboratory test conditions were adequate and had not changed significantly. The results of the most recent test are shown in Table 5 (attached).

PREPARATION OF THE TEST SOLUTION
- Test solutions for the range-finding and definitive tests were prepared by directly adding appropriate amounts of test item to dilution water and facilitating its dispersion by stirring for 30 minutes.
- The insoluble test substance was placed on the surface of the water and the test solution was colourless and clear.
- In the definitive test, stock solutions were prepared by adding an appropriate amount of test item to test medium in glass aspirator bottles of appropriate size.
- The mixing aspirator bottle was stoppered and the aqueous test substance mixture was stirred for 24 hours on a magnetic stirplate with a Teflon stirbar at room temperature (approximately 23 to 25 °C).
- The vortex height was set so it was at least 10 % of the static liquid height.
- Stirring was stopped after 24 hours and the test substance was placed on the surface of the water.
- The stock solution was allowed to stand for one hour at room temperature prior to removal of any undissolved test item by filtration through a 0.45 µm Millipore filter membrane (first approximate 100 mL discarded) to produce the 100 % v/v saturated stock solution of test item.
- Test solution preparation details are shown in the table below.

OBSERVATIONS AND EVALUATIONS
- Abnormal fish responses observed during the test (such as mortality, inactivity, abnormal swimming pattern) were recorded.
- Fish were considered dead if there was no visible gill movement and if touching of the caudal peduncle produced no reaction.

RANGE-FINDING TEST
- The range-finding test, carried out under static conditions, was conducted to determine the range of concentrations for the subsequent test.
- Groups of fish (5 per group) were exposed to test solutions with nominal concentrations of 1.0, 10.0 and 100 mg/L. Once control group was also included in the study using test water without test substance. Each test tank contained 3L of test solution and replicates were not used.
- test fish were chosen randomly and put into appropriate test solutions after the temperature had been adjusted to the required value. This procedure was conducted within 30 minutes.
- Photoperiod was 16 hours light daily (intensity 1000 lux to 1500 lux).
- Temperature was 22.9 to 23.3 °C.
- Oxygen concentration was 80 to 90 % of air saturation and no aeration took place.
- Feeding of fish did not take place.
- Test duration was 96 hours.
- Dead fish were removed at least once daily and discarded.
- Fish mortality was recorded at 24, 48, 72 and 96 hours. The maximum concentration causing no mortality (LC0) and the minimum concentration causing 100 % mortality (LC100) were then determined.

DEFINITIVE TEST
- The static method was adopted in the definitive test.
- Stability of the test solution was confirmed by results given in Table 3 (attached) and deviation was within 20 %.
- Based on the results of the range-finding test, concentrations of 10 %, 20 %, 40 %, 60 % and 100 % v/v saturated solution were assigned in the definitive test.
- A concurrent blank control was used in the test.
- No replicate were used for the control or treatment groups.
- The initial number of fish was ten for each group.
- Test fish were chosen randomly and put into different test solutions after the temperature had been adjusted to the required value. This procedure was conducted within 30 minutes.
- Photoperiod was 16 hours light daily (intensity 1000 lux to 1500 lux).
- Temperature was 23.1 to 23.5 °C.
- Oxygen concentration was 73 to 93 % of air saturation and no aeration took place.
- Feeding of fish did not take place.
- Fish mortality was recorded at 3, 6, 24, 48, 72 and 96 hours and observations on individual behaviour were performed.
- Measurements of pH, dissolved oxygen concentration and temperature were performed and recorded daily.

VALIDITY OF TEST
- A control group comprising the same number of fish as that exposed to each test concentration was placed into test water alone.
- With the conditions maintained as previously described, K2Cr2O7 was used as a positive control substance and the resulting LC50 (24 h) was determined to be 269 mg/L. Results of this investigation are given in Table 5 (attached).
- Fish loading was 0.7 to 0.8 g fish (wet weight) per litre of test medium.
- During the test period, the pH values of the control and test media were between 7.91 and 8.23.
- Dissolved oxygen values varied from 73 to 93 % of air saturation values.
- otal hardness was in the range 136 to 145 mg/L as CaCO3.
- Temperature was maintained in the range 23.1 to 23.5 °C.
- All fish in the control group were normal.
- The study met the acceptability criteria prescribed by the protocol ( pH 6.0 to 8.5; dissolved oxygen concentration > 60 % of air saturation value; total hardness 10 to 250 mg/L as CaCO3; temperature 23 ± 2 °C; LC50 (24 h) for K2CrO7 in the range 200 to 400 mg/L.

STABILITY OF TEST SOLUTION AND CHEMICAL ANALYSIS
- A standard stock solution (I) of the test substance (89.0 mg/L) was prepared by dissolving 0.0089 g of test item in 100 mL methanol.
- A standard solution (II) of 10.0 mg/L v/v was prepared by adding 5.618 mL of standard stock solution I (89.0 mg/L) to 50.0 mL of 4 % nitric acid solution.
- A standard stock solution (III) of 1.00 mg/L v/v was prepared by adding 5.00 mL of standard stock solution II (10.0 mg/L) to 50.0 mL of 4 % nitric acid solution.
- Working solutions were prepared by pipetting appropriate amounts of standard solution into 10.0 mL of 4 % nitric acid solution.
- Details of the working solutions are shown in the table below.

ICP-MS CONDITIONS
- Main operating parameters are shown in the table below.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.221 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.57 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limits 0.51 to 0.65 mg/L
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
0.798 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
ANALYTICAL METHOD FOR DETERMINATION OF TEST SUBSTANCE IN WATER
(1) CALIBRATION CURVE
- A series of standard solutions of concentration 0.05, 0.10, 0.20, 0.30 and 0.50 mg/L were determined using the ICP-MS conditions described previously.
- Based on the test result, a linear regression equation of A = 713141c + 28978 was obtained from the concentration and ICP-MS response values for boron where A represents values for boron (ICPS) and c is the concentration of test substance (mg/L) (see Figure 1, attached).
- Linearity for the concentration range 0.05 to 0.50 mg/L was good (r = 0.9834).
(2) RECOVERY TEST
- Recovery samples of concentration 0.10 mg/L were prepared by adding 5.00 mL of standard solution III (1.00 mg/L) to a total volume of 50.0 mL test water.
- Recovery samples of concentration 1.00 mg/L were prepared by adding 5.00 mL of standard solution II (10.0 mg/L) to a total volume of 50.0 mL test water.
- Recovery water samples of concentration 1.00 mg/L were analysed by diluting 5.009 mL to 50.0 mL v/v with 4 % nitric acid solution followed by ICP-MS determination.
- Recovery water samples of concentration 0.10 mg/L were directly analysed by ICP-MS.
- Measurements obtained from the recovery test are given in Table 2 (attached).
- The chromatograph from the recovery test is shown in Figure 3 (attached).
- The mean recovery rates were 96.3 % (1.00 mg/L) and 97.1 % (10.0 mg/L).
- Relative standard deviation was 2.72 % (1.00 mg/L) and 2.38 % (10.0 mg/L).
(3) DETECTION LIMIT
- The minimum amount of test material detected by ICP-MS was found to be 1.0 x 10E-10 g and the minimum quantitation detection concentration for the water sample was 0.01 mg/L.

ANALYSIS OF THE TEST SUBSTANCE IN TEST SOLUTIONS
- Results from analysis of test samples are given in Table 3 (attached) for the definitive test.
- The results indicated that the concentration of test substance was stable (within 80 % of the initial concentration) in the water during the test period.
- The static method used in the definitive test was therefore considered to be reasonable.

TEST CONDITIONS
- The pH, dissolved oxygen concentration, total hardness and temperature of the control and treatment groups during the definitive test are shown in Table 4 (attached).
- During the whole test period, the pH values of the control and test media were between 7.91 and 8.23, dissolved oxygen concentration values varied from 73 to 93 % of air saturation, total hardness was in the range 136 to 145 mg/L as CaCO3 and temperature was maintained in the range 23.1 to 23.5 °C.

MORTALITY AND EFFECTS
- Mortality data relating to the range-finding test are given in Table 6 (attached) and mortality data from the definitive test are shown in Table 7 (attached).
- Visual observations of behaviour and abnormalities during the definitive test are summarised in Table 8 (attached).
- During the test period, all fish in the control and 10 % v/v saturated solution were alive and appeared normal.
- Effects (fish lying on side or back) occurred at nominal test concentrations of 20 % stock solution and all fish were dead after 96 hours exposure at nominal test concentrations of 40 % stock solution.
Results with reference substance (positive control):
- The LC50 (24 h) for the postive control substance (K2Cr2O7) was determined to be 269 mg/L.
- Results of this investigation are given in Table 5 (attached).
Reported statistics and error estimates:
- The trimmed Spearman-Karber method (version 1.5; USEPA) was used to calculate LC50 values and confidence limits.
Validity criteria fulfilled:
yes
Conclusions:
The results showed that under valid static test conditions the LC50 (96 h) value was 0.57 mg/L (95 % confidence limits 0.51 to 0.65 mg/L based on measured concentration). The maximum tested concentration causing no mortality was 0.221 mg/L and the minimum concentration causing 100 % mortality was 0.798 mg/L.
Executive summary:

GUIDELINE

The acute toxicity of the test item to Gobiocypris rarus was investigated under static conditions in accordance with The Guidelines for the testing of chemicals (HJ/T 153-2004) and with reference to Procedure 203 of the Guidelines for Testing of Chemicals “Fish Acute Toxicity Test” (OECD; 1992).

 

METHODS

A range-finding test was performed followed by a definitive test. Nominal concentrations of 1.0, 10.0 and 100 mg/L were used in the range finding test and nominal concentrations of 10 %, 20 %, 40 %, 60 %, 80 % and 100 % v/v saturated solution were used in the definitive test. The stock solution was 100 % saturated solution prepared by dissolving 0.3001 g or 0.3002 g of test material in 3 L of test media and this was diluted to obtain 10 %, 20 %, 40 %, 60 % and 80 % v/v saturated solutions. Water samples taken from blank control and treatment groups in the definitive test were analysed. Five fish were used for the range-finding test (no replicates) and ten fish per treatment were used for the definitive test (no replicates). The fish were exposed to test item for 96 hours.

 

RESULTS

Concentrations of test substance were quantified using ICP-MS and the measured concentrations were 0.221 mg/L (10 %), 0.470 mg/L (20 %), 0.798 mg/L (40 %), 1.16 mg/L (60 %), 1.68 mg/L (80 %) and 2.00 mg/L (100 %). The analytical results showed that the concentration of test item was consistent in the test medium throughout the 96-hour study period (deviation within 20 %) and that a static procedure was reasonable. During the test period, all fish in the control and 10 % treatment group were alive and appeared normal. Effects (fish lying on side or back) occurred at nominal test concentrations of 20 % v/v saturated solution. All fish were dead after 96 hours exposure at a nominal test concentration of 40 % v/v saturated solution. A positive control substance (K2Cr2O7) assessed under the same conditions gave an LC50 (24 h) value of 269 mg/L. The study met the acceptability criteria prescribed by the protocol (pH 6.0 to 8.5; dissolved oxygen concentration > 60 % of the air saturation value; total hardness 10 to 250 mg/L as CaCO3; temperature 23 ± 2 °C; LC50 (24 h) for K2Cr2O7 in the range 200 to 400 mg/L).

 

CONCLUSION

The results showed that under valid static test conditions the LC50 (96 h) value was 0.57 mg/L (95 % confidence limits 0.51 to 0.65 mg/L based on measured concentration). The maximum tested concentration causing no mortality was 0.221 mg/L and the minimum concentration causing 100 % mortality was 0.798 mg/L.

Description of key information

In the key study, exposure of rainbow trout to the test item gave an LC50 (96 h) value of 1.3 mg/L (95 % confidence limits 1.1 to 1.6 mg/L) based on carbon content. The No Observed Effect Concentration (NOEC) based on carbon content was 1.1 mg/L and the Lowest Observed Effect Concentration (LOEC) based on carbon content was 1.6 mg/L. With respect to boron content, the LC50 (96 h) value was determined to be 8.4 mg/L (95 % confidence limits 6.4 to 11 mg/L). The No Observed Effect Concentration (NOEC) based on boron content was 6.4 mg/L and the Lowest Observed Effect Concentration (LOEC) based on boron content was 11 mg/L (OECD 203 and EU Method C.1). The results of a supporting study showed that under valid static test conditions the LC50 (96 h) value was 0.57 mg/L (95 % confidence limits 0.51 to 0.65 mg/L based on measured concentration). The maximum tested concentration causing no mortality was 0.221 mg/L and the minimum concentration causing 100 % mortality was 0.798 mg/L (OECD 203).

Key value for chemical safety assessment

LC50 for freshwater fish:
8.4 mg/L

Additional information

Key study

GUIDELINE

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203 “Fish, Acute Toxicity Test” referenced as Method C.1 of Commission Regulation (EC) No 440/2008.

 

METHODS

Due to the low aqueous solubility and pure nature of the test item, the test medium was prepared as a slow stir saturated solution for the purposes of the definitive test. Following a preliminary range-finding test, fish were exposed, in groups of seven, to the test item over a range of nominal concentrations (1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution) for a period of 96 hours at a temperature of 14 °C under semi-static conditions. The number of mortalities and any sub-lethal effects of exposure were determined in each test and control vessel at 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

RESULTS

Chemical analysis of the 5.6 and 10 % v/v saturated solution test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured boron concentrations of 0.13 to 0.24 mg/L (equivalent to test item concentration of 6.06 to 11.09 mg/L based on a test item boron content of 2.146 %). Total Organic Carbon (TOC) analysis of the 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) showed measured carbon concentrations of less than the limit of quantification of the analytical method (LOQ 1.0 mg C/L to 3.01 mg C/L; equivalent to test item concentrations of less than LOQ to 4.0 mg/L based on test item carbon content of 75.78 %).

 

CONCLUSION

Exposure of rainbow trout to the test item gave an LC50 (96 h) value of 1.3 mg/L (95 % confidence limits 1.1 to 1.6 mg/L) based on carbon content. The No Observed Effect Concentration (NOEC) based on carbon content was 1.1 mg/L and the Lowest Observed Effect Concentration (LOEC) based on carbon content was 1.6 mg/L. With respect to boron content, the LC50 (96 h) value was determined to be 8.4 mg/L (95 % confidence limits 6.4 to 11 mg/L). The No Observed Effect Concentration (NOEC) based on boron content was 6.4 mg/L and the Lowest Observed Effect Concentration (LOEC) based on boron content was 11 mg/L.

Supporting study

GUIDELINE

The acute toxicity of the test item to Gobiocypris rarus was investigated under static conditions in accordance with The Guidelines for the testing of chemicals (HJ/T 153-2004) and with reference to Procedure 203 of the Guidelines for Testing of Chemicals “Fish Acute Toxicity Test” (OECD; 1992).

 

METHODS

A range-finding test was performed followed by a definitive test. Nominal concentrations of 1.0, 10.0 and 100 mg/L were used in the range finding test and nominal concentrations of 10 %, 20 %, 40 %, 60 %, 80 % and 100 % v/v saturated solution were used in the definitive test. The stock solution was 100 % saturated solution prepared by dissolving 0.3001 g or 0.3002 g of test material in 3 L of test media and this was diluted to obtain 10 %, 20 %, 40 %, 60 % and 80 % v/v saturated solutions. Water samples taken from blank control and treatment groups in the definitive test were analysed. Five fish were used for the range-finding test (no replicates) and ten fish per treatment were used for the definitive test (no replicates). The fish were exposed to test item for 96 hours.

 

RESULTS

Concentrations of test substance were quantified using ICP-MS and the measured concentrations were 0.221 mg/L (10 %), 0.470 mg/L (20 %), 0.798 mg/L (40 %), 1.16 mg/L (60 %), 1.68 mg/L (80 %) and 2.00 mg/L (100 %). The analytical results showed that the concentration of test item was consistent in the test medium throughout the 96-hour study period (deviation within 20 %) and that a static procedure was reasonable. During the test period, all fish in the control and 10 % treatment group were alive and appeared normal. Effects (fish lying on side or back) occurred at nominal test concentrations of 20 % v/v saturated solution. All fish were dead after 96 hours exposure at a nominal test concentration of 40 % v/v saturated solution. A positive control substance (K2Cr2O7) assessed under the same conditions gave an LC50 (24 h) value of 269 mg/L. The study met the acceptability criteria prescribed by the protocol (pH 6.0 to 8.5; dissolved oxygen concentration > 60 % of the air saturation value; total hardness 10 to 250 mg/L as CaCO3; temperature 23 ± 2 °C; LC50 (24 h) for K2Cr2O7 in the range 200 to 400 mg/L).

 

CONCLUSION

The results showed that under valid static test conditions the LC50 (96 h) value was 0.57 mg/L (95 % confidence limits 0.51 to 0.65 mg/L based on measured concentration). The maximum tested concentration causing no mortality was 0.221 mg/L and the minimum concentration causing 100 % mortality was 0.798 mg/L.