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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:
15 September 2017 - 27 October 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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
FINAL TEST
Test concentrations:
- The test substance9: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
- Controls: Test medium without test item or other additives

SAMPLING METHOD
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h and t=96 h
Volume: 2.0 mL
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Additionally: reserve samples of 2.0 mL were taken from all test solutions for possible analysis.


Vehicle:
no
Details on test solutions:
PREPARATION OF TESTS SOLUTIONS
The batch of the test substance tested was a colourless to light yellow viscous liquid with a purity of 97%. The test item was not completely soluble in test medium at the initial concentrations prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to accelerate and ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of 1¾ hour or 1 hour (range-finder and final test, respectively). Thereafter, the aqueous Saturated Solution was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the Saturated Solution in test medium. The test solutions were colorless and increasingly hazy from 10% Saturated Solution onwards at the end of the preparation procedure.

CONTROLS:
Test medium without test substance or other additives
Test organisms (species):
Cyprinus carpio
Details on test organisms:
Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length: Range-finding test: 2.7 ± 0.2 cm; Final test: 2.4 ± 0.2 cm
Mean weight: Range-finding test: 0.23 ± 0.06 g; Final test: 0.17 ± 0.06 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 51

Quarantine/Acclimatisation : At least 12 days after delivery
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, 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

Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters: were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
180 mg CaCO3 per litre
Test temperature:
Final test: 20.5 - 21.9 °C
Temperature of culture medium was: 21.8°C.
pH:
Final test: 7.5 - 7.8
pH of culture medium was: 7.9
Dissolved oxygen:
Final test: 6.0 - 9.2 mg/L (oxygen > 60% of air saturation); amount of oxygen decreased during the test
Nominal and measured concentrations:
Analysis of the samples taken at the start and the end of the final test showed that measured concentrations for both peaks 1 and 2 were stable and in agreement with the expected nominal concentrations (between 10 and 100 mg/L). The range tested based on actual concentrations was 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
Test duration: 96 hours
Test type: Static
Test vessels: 3.5 litres, all-glass, containing 3 litres of test solution
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Number of fish: 7 per concentration
Number of vessel per concentration (replicates): 1
Number of vessel per control (replicates): 1

OTHER TEST CONDITIONS
Loading: 0.40 g fish/Litre, i.e. 7 fish per 3 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration: The test media were not aerated during the test.

Feeding: No feeding from 24 hours prior to the test and during the total test period
Introduction of fish: Within 24 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 1.0°C.

EFFECT PARAMETERS MEASURED
Mortality and other effects were measured and recorded: At 2, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.
Dissolved oxygen content, pH and temperature were measured and recorded: Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

MEDIUM
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, 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

CONCENTRATIONS FINAL TEST
The test substance: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.
Controls: Test medium without test item or other additives.

RANGE FINDING TEST
Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three fish per concentration
- concentration: w 1.0, 10 and 100% saturated solution prepared at a loading rate of 100 mg/L.
- Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration on days 0, 1, 3 and 4, whereas on day 2 test conditions in all test groups were measured.
- Samples for possible analysis: t = o h, 24 h, 96 h


Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (PCP)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
ca. 90 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Measured concentrations
Basis for effect:
mortality (fish)
Remarks on result:
other:
Remarks:
LC50 was already reached after 72 hours of exposure
Details on results:
RANGE FINDING TEST:
No mortality or clinical effects were observed up to and including 10% Saturated Solution, while all fish died in the undiluted 100% Saturated Solution, already after 48 h.
Based on these results samples taken from the solutions containing 10 and 100% Saturated Solutions were analysed. Analyses was based on the two major peaks present in the chromatograms, further referred to as peak1 and peak 2. Concentrations measured for peak 1 were stable and in agreement with the expected nominal concentrations (10 and 100 mg/L), whereas the concentrations measured for peak 2 were stable but at ~70% relative to what was expected based on the loading rates.
Hence, the expected LC50, when based on the lowest recovery for peak 2, was between 7 and 70 mg/L.

FINAL TEST:
The responses recorded in this test allowed for reliable determination of an LC50 and were in agreement with what was expected based on the results of the range-finding test. No clinical effects or mortality was observed up to and including 56 mg/L, while 6 of the 7 fish exposed to 100 mg/L died during the test period.
Clinical effects observed were limited to the highest test group scoring of day 1 when 4 fish were observed to be swimming hypo actively and 3 fish that showed loss of equilibrium

Analysis of the samples taken at the start and the end of the final test showed that measured concentrations for both peaks 1 and 2 were stable and in agreement with the expected nominal concentrations (between 10 and 100 mg/L). The range tested based on actual concentrations was 10, 18, 32, 56 and 100 mg/L.
Results with reference substance (positive control):
The 96h-LC50 for carp exposed to PCP was 0.32 mg/L (95 % confidence interval between 0.22 and 0.46 mg/L). This effect was already reached within 24 hours of exposure.
The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.
Sublethal observations / clinical signs:

Incidence of Mortality and Total Mortality During the Final Test

test substance
Concentration (mg/L)

Initial

Number
of fish

Cumulativemortality

Total

Mortality (%)

2h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

10

7

0

0

0

0

0

0

18

7

0

0

0

0

0

0

32

7

0

0

0

0

0

0

56

7

0

0

0

0

0

0

100

7

0

0

5

6

6

86

Final Test: Test Samples – based on Peak 1

Time of sampling

[hours]

Date of sampling

Date of
analysis1

Percentage of SS2

[%]

Analyzed test item concentration
[mg/L]

Relative to
initial

[%]

0

09 Oct 2017

26 Oct 2017

0

n.d.

0

 

 

 

10

10.4

 

 

 

 

18

18.8

 

 

 

 

32

35.3

 

 

 

 

56

59.6

 

 

 

 

100

104

 

96

13 Oct 2017

26 Oct 2017

0

n.d.

n.a.

 

 

 

10

11.4

110

 

 

 

18

20.6

109

 

 

 

32

37.7

107

 

 

 

56

63.5

107

 

 

 

100

108

104

1     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

n.d.Not detected.

n.a.Not applicable.

Final Test: Test Samples – based on Peak 2

Time of sampling

[hours]

Date of sampling

Date of
analysis1

Percentage of SS2

[%]

Analyzed test item concentration
[mg/L]

Relative to
initial

[%]

0

09 Oct 2017

26 Oct 2017

0

n.d.

 

 

 

 

10

9.04

 

 

 

 

18

16.6

 

 

 

 

32

30.4

 

 

 

 

56

51.2

 

 

 

 

100

90.1

 

96

13 Oct 2017

26 Oct 2017

0

n.d.

n.a.

 

 

 

10

9.12

101

 

 

 

18

17.1

103

 

 

 

32

29.9

98

 

 

 

56

51.3

100

 

 

 

100

88.1

98

1     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

n.d.Not detected.

n.a.Not applicable.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present static test, the 96h-LC50 of the test substance was estimated to correspond with 90 mg/L based on actual measured exposure concentrations and was already reached after 72 hours of exposure.
Executive summary:

A study was conducted to determine the acute toxicity of the test substanceto carp (Cyprinus carpio) under static conditions according to OECD Guideline 203.

The final test was performed based on the results of a preceding range-finding test. Seven fish per test group were exposed to an untreated control and to10, 18, 32, 56 and 100% of a Saturated Solution prepared at a loading rate of 100mg/L.Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test, showing that measured concentrations for both peaks 1 and 2 (the two major peaks present in the chromatograms) were stable and in agreement with the expected nominal concentrations.

No clinical effects or mortality was observed up to and including 56 mg/L, while 6 of the 7 fish exposed to 100 mg/L died during the test period. The 96h-LC50 of the test substance was estimated to correspond with 90 mg/L.

All criteria for acceptability of the test were met and the present toxicity study is classified as reliable without restrictions.

Description of key information

A short term toxicity study with the test substance was performed according to OECD guideline No. 203 and GLP principles.

The study was performed on carps, under static conditions, for 96 hours

No effects or mortality was observed up to and including 56 mg/L, while 6 of the 7 fish exposed to 100 mg/L died during the test period. The 96h-LC50 of the test substance was estimated to correspond with 90 mg/L.

ns.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
90 mg/L

Additional information

The acute toxicity of the test substance to fish (Cyprinus carpio) was investigated under static conditions according to OECD guideline 203.

- Range finding test: 1.0, 10, 100 mg/L

- final test: 0, 10, 18, 32, 56 and 100 mg/L, seven fishes for test group

The measured concentrations for both peaks 1 and 2 (the two major peaks present in the chromatograms) were stable and in agreement with the nominal concentrations.

No effects or mortality was observed up to and including 56 mg/L, while 6 of the 7 fish exposed to 100 mg/L died during the test period. The 96h-LC50 of the test substance was estimated to correspond with 90 mg/L.

All criteria for acceptability of the test were met and the present toxicity study is classified as reliable without restrictions.