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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 September 2017 to 03 November 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
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 18/07/2017 - 20/07/2017. Date of issue: 28/11/2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 440038-285
- Expiration date of the lot/batch: 01 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 ºC in the dark
- Stability under test conditions: stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control and test groups test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. Duplicate samples at 0 and 72 hours (fresh media), 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.
- Sampling method: no data available
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Preliminary media trial results:
Visual observations made on the centrifuged test samples showed they were hazy homogenous dispersions, as such the use of centrifugation as a means to remove the undissolved test item present was considered inappropriate. Whilst the filtered samples yielded results of less than the LOQ, determined to be 0.029 mg/L, this does not infer that no test item was in solution, just that any which was present was so at a concentration of less than 0.029 mg/L.
- Method: Based on the preliminary trial the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 liter discarded) to give a 100% v/v saturated solution of the test item. The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): precipitation
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): ' juvenile'
- Length at study initiation (length definition, mean, range and SD): mean standard length = 4.8 cm (SD = 0.25)
- Weight at study initiation (mean and range, SD): Mean body weight of 0.99 g (SD = 0.20)

ACCLIMATION
- Acclimation period: Fish were acclimatized to test conditions from 30 October 2017 to 06 November 2017.
- Acclimation conditions (same as test or not): Fish were maintained in a glass fiber tank with a "single pass" water renewal system. The water temperature was controlled at approximately 14 °C to 15 °C with a dissolved oxygen content of greater than or equal to 9.3 mg O2/L.
- Type and amount of food during acclimation: commercial trout pellets
- Feeding frequency during acclimation: no data
- Health during acclimation (any mortality observed): no mortality


FEEDING DURING TEST
Discontinued approximately 24 hours prior to the start of the definitive test’
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
140 mg/L as CaCO3
Test temperature:
14 to 16 °C
pH:
7.2-8.1
Dissolved oxygen:
8.3-10.8 mgO2/L
Salinity:
22.35 mg Na/L based on typical water characteristics for supply zone ZDB13
Conductivity:
345 μS/cm at 20°C based on typical water characteristics for supply zone ZDB13
Nominal and measured concentrations:
0, 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 to 30L glass exposure vessels
- Aeration: via narrow bore glass tubes
- Renewal rate of test solution (frequency/flow rate): daily (semi-static)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the 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) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
Typical Water Characteristics for Supply Zone ZDB13:

Analysis Typical Value Units
Hardness Level Slightly Hard N/A
Hardness clark 8.76 Degrees clark
Hardness french 12.52 French degrees
Hardness german 7.01 German degrees
Aluminium 7.46 mg Al/L
Chlorine 0.32 g/L
Coliform bacteria 0 no./100mL
Colour 0.6 mg/l Pt/Co
Conductivity 345 μS/cm at 20°C
E.Coli bacteria 0 no./100mL
Fluoride 0.24 mg F/L
Iron 19.9 μg Fe/L
Manganese 2.9 μg Mn/L
Nitrate 7.61 mg NO3/L
Odour 0 Dilution number
Pesticides 0 μg/L
pH 7.66 pH Value
Sodium 22.35 mg Na/L
Taste 0 Dilution number
Plumbing Metals
Copper 0.02 mg Cu/L
Lead 0.51 μg Pb/L
Nickel 3.1 μg Ni/L



OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: There were no sub-lethal effects of exposure during the range-finding test. Based on this information, a single test concentration of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
This study showed that there were no toxic effects at saturation.
Sublethal observations / clinical signs:

Range-finding Test:

Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1

Table 1: Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)

Cumultative Mortality (Initial Population = 3)

1 Hour

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

99.5 Hours

Control

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

There were no sub-lethal effects of exposure during the range-finding test.

Chemical analysis of the 100% v/v saturated solution fresh test solution at 0 hours and 24-Hour old test media showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed was obtained which was determined to be 0.0048 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ

Based on this information, a single test concentration of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

Definitive Test

Verification of Test Concentrations

Chemical analysis of the fresh test solutions at 0 and 72 hours and corresponding expired test media at 24 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0048 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Mortality Data

Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2.

Table 2: Cumulative Mortality Data in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)

Cumultative Mortality (Initial Population = 7)

Mortality (%)

1 Hour

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

96 Hours

96

Hours

Control

0

0

0

0

0

1*

1

14.3

100

0

0

0

0

0

0

0

0

= One fish found dead on bench top approximately 53 hours after start of exposure

There were no mortalities in 7 fish exposed to a test concentration of 100% v/v saturated solution for a period of 96 hours.

Sub-Lethal Effects

There were no sub-lethal effects of exposure observed in the test.

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 and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.

Observations on Test Item Solubility

The test item preparations were observed to be clear colorless solutions throughout the test.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.
This study showed that there were no toxic effects at saturation.
Executive summary:

SUMMARY

1. Introduction

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.

2. Methods

Information provided by the Sponsor indicated the water solubility of the test item to be 5.8 x 10-5 mg/L.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a saturated solution method of preparation was appropriate for this test item.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100% v/v saturated solution for a period of 96 hours at a temperature of approximately 14 ºC to 16 °C under static test conditions. The test item solution was prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a nominal concentration of 100% v/v saturated solution. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

3. Results

Chemical analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0048 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.

Description of key information

One key study is available. The study is performed in accordance with OECD TG 203 and under the conditions of GLP.

Key value for chemical safety assessment

Additional information

Chemical analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0048 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.