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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:
From 14 November 2015 to 08 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 7346-2 (Water quality – Determination of the acute lethal toxicity of items to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)])
Version / remarks:
(1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Not completely soluble in test medium at 100 mg/L
Analytical monitoring:
yes
Details on sampling:
- Concentrations sampled: all test concentrations;
-- At the start of the test (t=0h) and after 72 hours (t=72h) from the freshly prepared solutions
-- At the first renewal (t=24h) and at the end of the test (t=96h) from the 24-hour old solutions
- Volume: 2.0 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: in a freezer
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/L. A magnetic stirring period of two days was applied to ensure reaching maximum dissolution in test medium. The resulting dispersion was left to settle for one hour after which the clear and colourless Water Soluble Fraction (WSF) was collected. Lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Characteristics: F1 from a single parent-pair bred in UV-treated water
- Mean length at study initiation:
-- Range-finding test: 2.1 ± 0.1 cm
-- Final test: 2.2 ± 0.2 cm
- Mean weight at study initiation:
-- Range-finding test: 0.14 ± 0.05 g
-- Final test: 0.20 ± 0.08 g

ACCLIMATION
Acclimation period: At least 12 days after delivery

FEEDING
Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands).

FEEDING DURING TEST
No feeding for 24 hours prior to the test and during the total test period.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
180 mg CaCO3 per litre
Test temperature:
20.6 - 21.9 °C
pH:
7.4 - 7.8
Dissolved oxygen:
7.3 - 9.9 mg/L
Nominal and measured concentrations:
- 1.0, 1.8, 3.2, 5.6 and 10% of a WSF, prepared at a loading rate of 100 mg/L
- Calculated average exposure concentrations: 0.64, 1.2, 2.0, 3.5 and 7.0 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 3.5 litres, all-glass, containing 3 litres of test solution
- Aeration: The test media were not aerated during the test
- Renewal rate of test solution: daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.47 g fish/Litre, i.e. 7 fish per 3 litres of test medium

TEST MEDIUM
Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3, formulated using
RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCL: 4.2 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

RANGE FINDING STUDY (STATIC EXPOSURE):
- Test concentrations: 0.1, 1.0, 10 and 100% of a WSF prepared at a loading rate of 100 mg/L (3 fish/concentration)
- Results used to determine the conditions for the definitive study:
-- No mortality or clinical effects were observed in test groups representing 0.10 and 1.0% of the WSF
-- All fish died within 24 hours in the test groups representing 10 and 100% WSF
-- Based on these results, samples taken from 1.0 and 100% WSF were analysed. The initial measured concentrations were 0.74 and 73 mg/L, respectively for 1.0 and 100% WSF.
-- The concentration measured in the highest test group decreased only slightly to 66 mg/L during the test period. However, the concentration measured in the group containing 1.0% WSF decreased significantly to 0.34 mg/L.
-- The expected LC50 was therefore between actual exposure concentrations of 0.7 and 7 mg/L (based on initial exposure concentrations).
-- It was decided to continue testing with daily renewals as to maintain concentrations stable in the final study
Reference substance (positive control):
yes
Remarks:
(yearly sensitivity check using pentachlorophenol)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Reached already after 72 hours of exposure; 95% confidence interval: 1.2 - 2.0 mg/L
Details on results:
- Analysis of the samples taken during the final test showed that measured concentrations decreased slightly during the 24-hour renewal intervals. The average decreases ranged between 20 and 35% relative to the initial measured concentrations. Based on these results, the average exposure concentrations were calculated (see below). The range tested based on average measured concentrations corresponded with 0.64, 1.2, 2.0, 3.5 and 7.0 mg/L.
- Mortality data: see below
Results with reference substance (positive control):
In the latest sensitivity check, performed in the period 4 - 8 January 2016, a 96h-LC50 of 0.24 mg/L was determined for carp exposed to pentachlorophenol. This is within the range observed during the past years.

Measured concentrations: 

Test group

(%WSF)

Measured concentration (mg/L)

Average

(mg/L)

t=0h

t=24h

t=72h

t=96h

1.0

0.765

0.547

0.725

0.541

0.64

1.8

1.33

0.957

1.37

1.03

1.2

3.2

2.45

1.58

n.d.

n.d.

2.0

5.6

4.20

2.84

n.d.

n.d.

3.5

10

7.69

6.29

n.d

n.d.

7.0

n.d.: not determined (fish had died within 24 hours)

Incidence of mortality and total mortality during the final test:

Concentration

(mg/L)

Initial number of fish

Cumulative mortality

Total mortality

(%)

3¾h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

0.64

7

0

0

0

0

0

0

1.2

7

0

0

0

0

0

0

2.0

7

0

0

0

7

7

100

3.5

7

0

4

6

7

7

100

7.0

7

0

7

7

7

7

100

 

Validity criteria fulfilled:
yes
Conclusions:
In a study performed in accordance with OECD 203 (1992) and according to GLP principles, an 96h-LC50 of 1.5 mg/L was determined, based on average measured exposure concentrations.
Executive summary:

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the substance to carp was investigated. As the substance was not completely soluble in test medium at 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L. A magnetic stirring period of two days was applied to ensure reaching maximum dissolution in test medium. The resulting dispersion was left to settle for one hour after which the clear and colourless Water Soluble Fraction (WSF) was collected. Lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. A semi-static final test was performed based on the results of a preceding static combined limit/rangefinding test. Seven carp per test group were exposed to an untreated control and to test groups representing 1.0, 1.8, 3.2, 5.6 and 10% of a WSF prepared at a loading rate of 100 mg/L. Test solutions were daily renewed during the 96-hour test period. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at t=0h and t=72h, while 24-hour old solutions were sampled at t=24h and t=96h. Analysis of the samples taken during the final test showed that measured concentrations decreased slightly during the 24-hour renewal intervals. The average decreases ranged between 20 and 35% relative to the initial measured concentrations. Based on these results, the average exposure concentrations were calculated to be 0.64, 1.2, 2.0, 3.5 and 7.0 mg/L. The study met the acceptability criteria. Based on the results of the test, an 96h-LC50 of 1.5 mg/L was determined based on average measured exposure concentrations (95% confidence interval between 1.2 and 2.0 mg/L) and was already reached after 72 hours of exposure.

Description of key information

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, an 96h-LC50 of 1.5 mg/L was determined, based on average measured exposure concentrations.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.5 mg/L

Additional information

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the substance to carp was investigated. As the substance was not completely soluble in test medium at 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L. A magnetic stirring period of two days was applied to ensure reaching maximum dissolution in test medium. The resulting dispersion was left to settle for one hour after which the clear and colourless Water Soluble Fraction (WSF) was collected. Lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. A semi-static final test was performed based on the results of a preceding static combined limit/rangefinding test. Seven carp per test group were exposed to an untreated control and to test groups representing 1.0, 1.8, 3.2, 5.6 and 10% of a WSF prepared at a loading rate of 100 mg/L. Test solutions were daily renewed during the 96-hour test period. Samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at t=0h and t=72h, while 24-hour old solutions were sampled at t=24h and t=96h. Analysis of the samples taken during the final test showed that measured concentrations decreased slightly during the 24-hour renewal intervals. The average decreases ranged between 20 and 35% relative to the initial measured concentrations. Based on these results, the average exposure concentrations were calculated to be 0.64, 1.2, 2.0, 3.5 and 7.0 mg/L. The study met the acceptability criteria. Based on the results of the test, an 96h-LC50 of 1.5 mg/L was determined based on average measured exposure concentrations (95% confidence interval between 1.2 and 2.0 mg/L) and was already reached after 72 hours of exposure.