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Ecotoxicological information

Short-term toxicity to fish

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Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 21 February 2001 and 9 march 2001.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with agreed test protocols following OECD Guidelines without deviations. Acceptable restrictions such as lack of test material analyses are not expected to have a major impact on the test result.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: liquid at room temperature
- Analytical purity: not provided.
- Stability under test conditions: stable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
No data available.

Test solutions

Vehicle:
no
Details on test solutions:
Five water accommodated fractions (WAF) were prepared by direct addition of weighed test substance to 21 L of test water to give nominal concentrations of:  3.2, 5.6, 10, 18 and 32 mg/L.  Each mixture was stirred, by magnetic stirrer, for 24 hours then allowed to settle for 2 hours. The water phase was siphoned through a glass tube containing a glass wool plug (discarding the first 75-100 mL) to produce a WAF with little, or no insoluble residues.  The glass wool was pre-conditioned by soaking overnight in deionized, reverse osmosis water, rinsing 3X and autoclaving.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow Trout
- Source: Fish were obtained from Donnington Fish Farm, Upper Swell, Gloucestershire, UK and maintained in-house.
- Age at study initiation (mean and range, SD): Juvenile (no further data available)
- Length at study initiation (length definition, mean, range and SD): 4.2 cm (sd = 0.1) at study end.
- Weight at study initiation (mean and range, SD): 0.70 g (sd = 0.15) at study end.
- Method of breeding: not specified
- Feeding during test: feeding discontinued ca. 24 h prior to start of definitive test.


ACCLIMATION
- Acclimation period: from 19 February 2001 to 5 March 2001.
- Acclimation conditions (same as test or not): as test
- Type and amount of food: commercial trout pellets
- Feeding frequency: ad lib on a daily basis
- Health during acclimation (any mortality observed): zero mortality in the 7 days prior to the start of the test.


QUARANTINE (wild caught): Not applicable.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Mortalities and sub-lethal effects were recorded at 3, 6, 24, 48, 72 and 96 hours.

Test conditions

Hardness:
Approximately 100 mg/L as CaCO3
Test temperature:
Approximately 14 oC
pH:
7.6-7.7
Dissolved oxygen:
7.8-10.3 mg/L (control) and 7.8-10.2 mg/L (test).
Salinity:
Not applicable, it is a freshwater test.
Nominal and measured concentrations:
Nominal concentrations: 3.2, 5.6, 10, 18 and 32 mg/L were used in the definitive test.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): covered
- Material, size, headspace, fill volume: 20Litre
- Aeration: yes, via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable.
- Renewal rate of test solution (frequency/flow rate): not applicable.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates):1
- No. of vessels per control (replicates):1
- No. of vessels per vehicle control (replicates):N/A
- Biomass loading rate: Based on the mean weight value this gave a loading rate of 0.35 g bodyweighvlitre.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 then 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 dechlorination and softening
- Total organic carbon: 2 mg C/1
- Particulate matter: 0.2 mg/l.
- Metals: various present, none above prescribed concentration values
- Pesticides: none above prescribed concentration values
- Chlorine: 0.16 mg/l
- Alkalinity: 92 mg/l
- Ca/mg ratio: 5
- Conductivity: 1500 uS/cm
- Culture medium different from test medium: Same
- Intervals of water quality measurement: no data available.


OTHER TEST CONDITIONS
- Adjustment of pH: no data available.
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: no data available.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and sub-lethal effects
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
ca. 24 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accomodated fraction
Basis for effect:
mortality
Remarks on result:
other: 18-32mg/L WAF
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accomodated fraction
Basis for effect:
mortality
Remarks on result:
other: 3.2 mg/l loading rate WAF
Details on results:
- Behavioural abnormalities: Sub-lethal effects of exposure were observed at the 5.6 mg/l loading rate WAF and above. These responses were increased pigmentation, loss of equilibrium and swimming at the bottom of the test vessels.
- Observations on body length and weight: details given in “Test species”
- Other biological observations: not applicable.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data available.
- Effect concentrations exceeding solubility of substance in test medium: not applicable.
Results with reference substance (positive control):
No data available.
Reported statistics and error estimates:
The LL50 values and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method using the ToxCalc computer software
Package, and at 48, 72 and 96 hours the LL50 values were calculated using the geometric mean method as follows:
LL50 value = SQRT (L1 x L2)
Where:
L1 = loading rate showing 0% mortality
L2 = loading rate showing 100 % mortality
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
If there are no mortalities between 0% and 100% mortality, then the geometric mean of the highest loading rate showing no lethality and the lowest loading rate showing 100% lethality is calculated. The loading rates resulting in 0% and 100% mortality will be the 95% confidence limits.
An estimate of the LL50 values at 3 and 6 hours was given by inspection of the mortality data.

Any other information on results incl. tables

Range-finding Studies

Cumulative mortality data from the exposure of rainbow trout to the test material during the range-finding studies showed no mortalities or sub lethal effects at the 3.2 mg/l loading rate WAF. However, mortalities or sub-lethal effects were observed at the 10,32, 100 and 1000 mg/l loading rate WAFs.

Based on this information loading rates of 3.2, 5.6, 10, 18 and 32 mg/l were prepared for the definitive study employing a single phase separation method. The WAFs were removed by siphon through Tygon tubing containing a glass wool plug (2 - 4 cm length) to remove any micro-dispersions which may have entered the WAF.

Definitive Studies

 

Cumulative Mortality Data

mg/L

WAF

3 hours

6 hours

24 hours

48 hours

72 hours

96 hours

Control

0

0

0

0

0

0

  3.2

0

0

0

0

0

0

  5.6

0

0

0a

0a

0a

0a

10

0

0

0a

0a

0a

0a

18

0a

0a

0ab

0ab

0ab

0ab

32

0a

0a

6bc

10

10

10

aincreased pigmentation

bincreased pigmentation and swimming at bottom

cincreased pigmentation, swimming at bottom and loss of equilibrium

Sub-lethal effects were observed at 5.6mg/L WAF and above.  Sub-lethal responses included increased pigmentation, loss of equilibrium and swimming at the bottom the test vessels.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The criteria for a valid test requiring mortality of the control not to exceed 10% and the maintenance of exposure conditions were met.
Conclusions:
The 96 h LL (Lethal Loading Rate) 50 value of the substance was determined (based on nominal loading rates) to be 24 mg/l loading rate WAF with 95% confidence limits of 18 - 32 mg/l loading rate WAF. The No Observed Effect Loading rate was 3.2 mg/l loading rate WAF.
Executive summary:

Introduction.A study was performed to assess the acute toxicity of the test material to rainbow trout(Oncorhynchusmykiss).The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.l of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), US CFR Title 40, Part 797, Section 1400 andUS EPADraft Ecological Effects Test Guideline OPPTS 850.1075.

Methods.Following a preliminary range-finding study, fish were exposed, in groups of ten, to Water Accommodated Fractions (WAFS) of the test material, prepared using a method employing a single phase separation over a range of nominal loading rates of 3.2, 5.6, 10, 18 and 32 mg/l for a period of 96 hours under static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination after 96 hours.

Results.The 96-Hour LL~ob*a sed on nominal loading rates was 24 mg/l loading rate WAF with 95% confidence limits of 18 - 32 mg/l loading rate WAF. The No Observed Effect Loading rate was 3.2 mg/l loading rateWAF.