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Diss Factsheets

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th February 2009 to 23rd June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US Code of Federal Regulations, Title 40, Part 797, Section 1600
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
Sponsor's identification: Experimental additive 20735-35

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test material in the test preparations were verified by chemical analysis (replicates pooled) on Days 0 (fresh media), 1, 5, 8, 12, 15, 19, 22, 26, 29 (old and fresh media) and 33 (old media) .

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test material has a low solubility in water and recognised auxiliary solvents and was therefore prepared as a saturated solution. An amount of test material (1125 mg) was dispensed onto the surface of 22.5 litres of dechlorinated tap water and stirred using a propeller stirrer at approximately 1500 rpm at approximately 25°C for 24 hours. After the stirring period the undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter to give a saturated solution with a nominal test concentration of 0.030 mg/L. Aliquots (100, 320, 1000 and 3200 mL) of the 0.030 mg/L test concentration were each dispersed in a final volume of 10 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the remainder of the test series of 0.00030, 0.00096, 0.0030 and 0.0096 mg/L respectively.
- Eluate: N/A
- Differential loading: N/A
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle: Dechlorinated tap water was used as the vehicle.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Strain: Pimephales promelas
- Source: The in-house breeding stock fish originated from fish that hatched on 2 February 2009 at Harlan Laboratories Ltd., Shardlow, Derbyshire, UK.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Not specified
- Method of collection of fertilised eggs: Fertilised eggs were collected from the breeding tanks on 21 May 2009 and used for the definitive test. The eggs were less than 24 hours old on introduction into the test system.
- Subsequent handling of eggs: Not specified


POST-HATCH FEEDING
- Start date: The larvae were fed from Day 5 of the study.
- Type/source of feed: The breeding stock were fed ZM 400 flake food daily. The larvae were fed protozoan (Paramecia micronucleatum) only from Day 5 to Day 7 as the larvae were too small at this time to feed on brine shrimp nauplii. On Day 8 and throughout the remainder of the test the larvae were fed brine shrimp nauplii only.
- Amount given: Not specified
- Frequency of feeding: Not specified.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Remarks on exposure duration:
Total study period 33 days; post-hatch period 28 days.
Post exposure observation period:
Not performed

Test conditions

Hardness:
The water hardness values were observed to range from 90 to 94 mg/L as CaCO3 at the start of the test and from 130 to 138 mg/L as CaCO3 at termination of the test.
The water hardness on Day 0 was low. This was a factor of the water supply to the laboratory at the time and could not be altered. Facility records showed that this low water hardness occurred for the first six days of the test. After this time the water hardness was within the 110 to 160 mg/L as CaCO3 acceptance limits for the remainder of the test.
Test temperature:
Temperature was maintained at 22°C to 25°C throughout the test.
pH:
There were no treatment related differences for pH during the test. The pH measured throughout the study varied from 7.8 to 8.6.
Dissolved oxygen:
The dissolved oxygen content was greater than or equal to 8.1 mg O2/L.
There were no treatment related differences for oxygen concentration during the test.
Salinity:
Not measured
Nominal and measured concentrations:
Nominal concentrations during the definitive test: 0.00030, 0.00096, 0.0030, 0.0096 and 0.030 mg/L.
Each test concentration was prepared in duplicate.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Not specified
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: For the definitive test glass beakers containing 2.5 litres of test media were used for each control and test vessel. At the start of the test 30 eggs were placed in an egg basket (glass tube approximately 5 cm diameter and 8 cm in height and fitted with a stainless steel mesh to retain the eggs). The egg baskets were then oscillated by a variable speed rocker arm system (see Figure 1) at a rate of approximately 1 oscillation per 2 minutes to induce non-turbulent flow of water through the egg baskets.
- Aeration: The test vessels received no auxiliary aeration. The diluent supply only was aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not specified
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of fertilized eggs/embryos per vessel: 30 fertilised eggs were introduced per concentration
- No. of vessels per concentration (replicates): 2 replicates
- No. of vessels per control (replicates): N/A
- No. of vessels per vehicle control (replicates): 2 replicates
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water was laboratory tap water dechlorinated by passage through an activated carbon filter (Elga AC1) and partly softened (Elga Nimbus 1248D Duplex Water Softener). After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 1.041 mg/L
- Particulate matter: Not specified
- Metals: Various metals of varying concentrations but would appear to be - Pesticides: 0 µg/L
- Chlorine: 0.297 mg/L
- Alkalinity: pH 7.26-8.24
- Ca/mg ratio: Not specified
- Conductivity: 421.385 µS/cm at 20°C
- Salinity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH, dissolved oxygen concentration (in mg/L and as a percentage of theair saturation value), temperature and light intensity were measured on a daily basis and before and after media renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: Not specified. The pH measured throughout the study varied from 7.8 to 8.6.
- Photoperiod: 16 hour light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: The light intensity measured throughout the study varied from ~300 Lux to ~800 Lux.

EFFECT PARAMETERS MEASURED: The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily. The criteria of death for eggs were marked loss of translucency and change in coloration leading to a white opaque appearance. The criteria of death for larvae and juvenile fish were one or more of the following: immobility, absence of respiratory movement, absence of heart beat, white opaque coloration and lack of reaction to mechanical stimulus. At test termination the length, wet weight and dry weight of the surviving fish were measured.

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
A range finding study was not performed.
A media preparation trial was conducted in order to determine the solubility of the test material under test conditions.
An amount of test material (550 mg) was dispersed in 11 litres of dechlorinated tap water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 25°C for 24 hours. The results of the media preparation trial showed a concentration of approximately 0.030 mg/L could be achieved using a saturated solution method of preparation. Given the low measured concentration it was considered unnecessary to conduct a range-finding test and the test concentrations for the definitive test were set at 0.00030, 0.00096, 0.0030, 0.0096 and 0.030 mg/L.

POST-HATCH DETAILS
- Begin of post-hatch period: The start of hatching was observed on Day 3 of the test and completion of hatching on Day 5.
- No. of hatched eggs (alevins)/treatment released to the test chamber: Not specified
- Release of alevins from incubation cups to test chamber on day no.: Not specified


FERTILIZATION SUCCESS STUDY
- Number of eggs used: 30 fertilised eggs were introduced per concentration
- Removal of eggs to check the embryonic development on day no.: Not specified
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
> 0.03 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fish length and dry weight
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.03 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: fish length and dry weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: The number of dead eggs and larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed. The mean survival rate of the larvae for the control group was 92% thereby satisfying the validation criterion for post-hatch survival success rate of greater than 70%. The mean survival rates for the test concentrations ranged between 92% and 97%. There were no significant mortalities or sub-lethal effects of exposure observed in any of the test concentrations.
- Days to hatch or time to release of young: The start of egg hatching was observed to be on Day 3 of the test and completion of hatching was observed on Day 5 of the test.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: The mean hatching rate for the control group was 95% thereby satisfying the validation criterion of greater than 66% hatching rate. The mean hatching rates for the test groups ranged from 90% to 100%.
- Number of fish in swim-up stage at one or more time periods: Not specified
- Observations on body length and weight of young and/or exposed parents at one or more time periods: Statistical analysis of the length data by analysis of variance showed the 0.00030, 0.00096, 0.0030, 0.0096 and 0.030 mg/L test concentrations not to be significantly different (P>=0.05) from the control group. Statistical analysis of the dry weight data showed no significant differences (P>=0.05) between the control group and 0.00030, 0.00096, 0.0030, 0.0096 and 0.030 mg/L test concentrations. Given this information and data assessment above it was considered that no effect on survival or growth attributable to the test material was observed.
- Number of healthy fish at end of test: The mean survival rate varied from 92-97%. By Day 33 of the study, 26-30 hatched larvae were still alive.
- Type of and number with morphological abnormalities: No sub-lethal effects of exposure observed in any of the test concentrations.
- Type of and number with behavioural abnormalities: No sub-lethal effects of exposure observed in any of the test concentrations.
- Type and number of developmental effects: No sub-lethal effects of exposure observed in any of the test concentrations.
- Type and magnitude of hormonal changes: No sub-lethal effects of exposure observed in any of the test concentrations.
- Other biological observations: No sub-lethal effects of exposure observed in any of the test concentrations.
- Effect concentrations exceeding solubility of substance in test medium: N/A. Pre-study solubility work showed the test material to have low solubility in water and recognised auxiliary solvents. Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions. However, the test preparations were observed to be clear, colourless solutions by visual inspection throughout the duration of the test.
- Incidents in the course of the test which might have influenced the results: Due to a technical error the data logger did not record the temperature for approximately 16 hours. This was considered not to affect the study given that no adverse effects were observed. Some of the temperatures during the definitive test were observed to be slightly below the range given in the protocol of 25 ± 2°C. This deviation was considered not to have affected the outcome or the validity of the test as the control larvae during testing showed no sub-lethal effects or mortality over the test period.
On several occasions the measured concentration was outside the 80% to 120% acceptance limits. However, given that the overall mean measured concentration was 100% of nominal, this was considered not to affect the study and therefore the results are based on nominal test concentrations only.
Results with reference substance (positive control):
Not appliable
Reported statistics and error estimates:
Statistical analysis of these data showed there were no significant differences (P>=0.05) between the control and all the test groups in terms of fish length or weight.

Any other information on results incl. tables

Analysis of the freshly prepared media showed measured concentrations to range from 57% to 151% of nominal. Analysis of the old media showed measured concentrations to range from 76% to 657% of nominal. On several occasions the measured concentration was outside the 80% to 120% acceptance limits. However, given that the overall mean measured concentration was 100% of nominal, this was considered not to affect the study and therefore the results are based on nominal test concentrations only.

Throughout the study a response was seen in control test samples and laboratory synthetic controls. This was generally a low level response which was not always quantifiable. Where the laboratory synthetic control was quantified, this response was subtracted from the test sample responses for that analysis. The interference showed the same chromatographic characteristics as the test material. This was considered to possibly be due to contamination although every effort was taken to minimise any cross contamination which may be detected by the highly sensitive instrument technique.

Table 1: Number of Dead Eggs in the Definitive Test

Nominal concentration (mg/L)

Number of Dead Eggs (Initial Population = 30)

Days

Total

1

2

3

4

5*

Control

R1

1

0

0

0

1

2

R2

1

0

0

0

0

1

0.00030

R1

0

0

0

0

0

0

R2

0

0

0

0

0

0

0.00096

R1

0

0

0

0

0

0

R2

1

0

0

0

0

1

0.0030

R1

1

0

0

0

0

1

R2

0

0

0

0

0

0

0.0096

R1

1

0

0

0

0

1

R2

2

0

0

0

0

2

0.03

R1

4

0

0

0

0

4

R2

0

0

0

0

0

0

* All surviving eggs hatched by day 5

R1-R2 = replicates 1 and 2

Table 2: Hatching and Survival Rates in the Definitive Test

Nominal concentration (mg/L)

Hatching rate (%)

Mean hatching rate (%)

Survival rate (%)

Mean survival rate (%)

Control

R1

93

95

86

92

R2

97

97

0.00030

R1

100

100

97

92

R2

100

87

0.00096

R1

100

99

93

92

R2

97

90

0.0030

R1

97

99

90

95

R2

100

100

0.0096

R1

97

95

97

95

R2

93

93

0.03

R1

87

90

100

97

R2

93

93

Table 3: Fish length and dry weight data obtained at termination of the test

-

Nominal Test Concentration (mg/L)

Control

0.00030

0.00096

0.0030

0.0096

0.030

Fish length (mean ±

Standard deviation, mm)

16.1±2.1

16.2±2.0

16.2±1.7

15.7±2.0

16.1±1.9

15.2±2.2

Dry Weight (mean ± Standard deviation, mg)

12.2±5.5

12.2±5.4

12.1±4.5

11.0±4.9

11.9±5.0

10.6±5.2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The application of the test material to newly laid eggs of fathead minnows was considered to have no effect on the survival or growth of the larvae.
The LOEC, based on nominal test concentrations, was considered to be greater than 0.030 mg/L on the basis that there were no significant differences (P>=0.05) in terms of fish length and dry weight when compared to the control at the end of the test.
The NOEC, based on nominal test concentrations, was considered to be 0.030 mg/L on the basis that there were no significant differences (P>=0.05) in terms of fish length and dry weight when compared to the control at the end of the test.
Executive summary:

A study was performed to assess the effects of the test material on freshly hatched larvae of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 210, "Fish, Early­Life Stage Toxicity Test", US Code of Federal Regulations, Title 40, Part 797, Section 1600 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1400.

Over the duration of the test there were no significant mortalities or sub-lethal effects of exposure resulting from the exposure of fathead minnow (Pimephales promelas) larvae to test concentrations of 0.00030, 0.00096, 0.0030, 0.0096 and 0.030 mg/L. The mean hatching rate for the control was 95% and the mean survival rate was 92%. The mean hatching rates for the test groups ranged from 90% to 100% and the mean survival rates from 92% to 97%. The NOEC was 0.030 mg/L.