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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 25 June, 2012 to 20 Aug, 2012
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:
yes
Remarks:
Inadvertently, fish were not observed for mortality and severe effects in the afternoon of nominal Day 1 during the final test.
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
Inadvertently, fish were not observed for mortality and severe effects in the afternoon of nominal Day 1 during the final test.
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)
Deviations:
yes
Remarks:
Inadvertently, fish were not observed for mortality and severe effects in the afternoon of nominal Day 1 during the final test.
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, December 14, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations with surviving fish and the control at the start and the end of the test. In addition, samples were taken from the four highest test concentrations after 24 h of exposure because all fish were found dead.

Volume: 2 mL
Storage: Samples were stored in a freezer until analysis.

Additionally, reserve samples of 2 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
DPHA a UVCB substance, was not completely soluble in test medium at the loading rates initially prepared. Weighing of test substance was performed under either dimmed or yellow light. Preparation of test solutions was performed under dimmed light conditions.

All test concentrations were prepared separately applying 2 d of magnetic stirring in the dark to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize for 3-3.5 h where after the Water Accommodated Fractions (WAFs) were siphoned off and used as test concentrations. WAFs prepared for both the range-finding test and the final test were observed to range from clear and colourless to hazy, while all WAFs prepared for the first LC50 test were observed to be clear and colourless.
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: 3.1 ± 0.3 cm
First LC50 test: 3.0 ± 0.2 cm
Final test: 2.6 ± 0.1 cm

Mean weight:
Range-finding test: 0.77 ± 0.31 g
First LC50 test: 0.81 ± 0.09 g
Final test: 0.47 ± 0.07 g

Feeding: No feeding from 24 h prior to the test and during the total test period
Reason for selection: This system has been selected as an internationally accepted species
Quarantine/Acclimatisation: At least 12 d after delivery
Feeding: Daily with pelleted fish food
Validity of batch: In the batch of fish used for the test, mortality during the 7 dprior 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:
hardness of 180 mg CaCO3/L
Test temperature:
Between 21.9 and 22.4 °C
pH:
Between 7.3 and 8.0
Dissolved oxygen:
Between 7.1 and 9.3
Nominal and measured concentrations:
Nominal concentration: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
measured concentrations: The quantitative analysis of the test substance in water was based on the response of the two major components DPPA and DPHA. Samples taken from the WAFs prepared at loading rates of 10 and 18 mg/L were analysed. The initial concentrations were 9.0 mg/L (DPPA) and 7.2 mg/L (DPHA) at the 10 mg/L WAF and 16 mg/L (DPPA) and 11 mg/L (DPHA) at the 18 mg/L WAF. These concentrations remained stable during the test period (102-105% of initial for DPPA and 87-94% of initial for DPHA at the end of the test). Given these results, the effect parameters could be based on initial exposure concentrations.Following a worst case scenario, DPHA concentrations were used to calculate the effect parameters, i.e. 7.2 and 11 mg/L.
Details on test conditions:
Test vessels: 20 and 40 L, all-glass, containing 15 L of test solution
Number of fish: 7 per concentration
Loading: 0.22 g fish/L, i.e. 7 fish per 15 L of test medium
Illumination: 16 h photoperiod daily using dimmed light
Aeration: The test media were not aerated during the test.
Dissolved oxygen content, pH and temperature measurement: Daily in all vessels with surviving fish, beginning at the start of the test
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
Effect parameters: 96 h LC0, 24, 48, 72 and 96h LC50

Reference substance (positive control):
yes
Remarks:
pentachlorophenol (PCP)
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
ca. 7.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
ca. 8.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 95% confidence interval 7.2-11 mg/L
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
ca. 13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: 95% confidence interval between 10 and 18 mg/L
Details on results:
Behavioral abnormalities:
- Mortality of control: No mortality in control
- Abnormal responses: No abnormalities were observed
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Under the conditions of the present test with carp exposed to PCP, the 96 h LC50 was 0.25 mg/L based on nominal concentrations, with a 95% confidence interval between 0.20 and 0.41 mg/L. This effect was already reached within 24 h 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.

Reported statistics and error estimates:
The LC50 could not be determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition). This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Sublethal observations / clinical signs:

Table. Incidence of mortality and total mortality during the final test:

Loading rate1

DPHA

(mg/L)

Initial

number

of fish

Cumulative mortality

Total

mortality (%)

2.45 h

24 h

48 h

72 h

96 h

Control

7

0

0

0

0

0

0

10 (7.2)

7

0

0

0

0

0

0

11

7

0

7

7

7

7

100

32

7

0

7

7

7

7

100

56

7

0

7

7

7

7

100

100

7

0

7

7

7

7

100

1WAF prepared at the given loading rate

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 96 h LC50 for the test substance was determined to be 8.9 mg/L (measured initial, WAF).
Executive summary:

A study was conducted to assess the acute toxicity of the test substance, DPHA to Cyprinus carpio in accordance with OECD Guideline 203, EU Method C.1 and ISO International Standard 7346-1, in compliance with GLP. Seven fish per concentration were exposed to Water Accommodated Fractions (WAFs) prepared at loading rates of 0, 10, 18, 32, 56 and 100 mg/L. The total test period was 96 h and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. The quantitative analysis of the test substance in water was based on the response of the two major constituents, DPHA and DPPA. Samples taken from the WAFs prepared at loading rates of 10 and 18 mg/L were analyzed. The initial concentrations were 9.0 mg/L (DPPA) and 7.2 mg/L (DPHA) at the 10 mg/L WAF and 16 mg/L (DPPA) and 11 mg/L (DPHA) at the 18 mg/L WAF. These concentrations remained stable during the test period (102-105% of initial for DPPA and 87-94% of initial for DPHA at the end of the test). The test substance induced no lethal effects in carp at a loading rate of 10 mg/L (LL0) (equivalent to 7.2 mg/L of the constituent DPHA (LC0)). Overall, the deviation between the nominal loading levels and measured concentrations was less than 20%. Given these results, the effect parameters could be based on initial measured concentrations or even based on the initial loading rates (WAF). In absence of information on the actual moiety driving the toxicity, it was decided to base the effect concentrations on the initial loading rates. Based on nominal loading rate, the 96 h LL50 was 13 mg/L (equivalent to the measured concentration of 8.9 mg/L) (Bouwman, 2013).

Description of key information

In the short term toxicity study to fish, the initial measured concentrations were 9.0 mg/L (DPPA) and 7.2 mg/L (DPHA) at the 10 mg/L WAF and 16 mg/L (DPPA) and 11 mg/L (DPHA) at the 18 mg/L WAF. These concentrations remained stable during the test period (102-105% of initial for DPPA and 87-94% of initial for DPHA at the end of the test). Overall, the deviation between the nominal loading levels and measured concentrations was less than 20%. Given these results, the effect parameters could be based on initial measured concentrations or even based on the initial loading rates (WAF). In absence of information on the actual moiety driving the toxicity, it was decided to base the effect concentrations on the initial loading rates. Based on nominal loading rate, the 96 h LL50 was 13 mg/L (equivalent to the measured concentration of 8.9 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
8.9 mg/L

Additional information

A study was conducted to assess the acute toxicity of the test substance, DPHA to Cyprinus carpio in accordance with OECD Guideline 203, EU Method C.1 and ISO International Standard 7346-1, in compliance with GLP. Seven fish per concentration were exposed to Water Accommodated Fractions (WAFs) prepared at loading rates of 0, 10, 18, 32, 56 and 100 mg/L. The total test period was 96 h and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. The quantitative analysis of the test substance in water was based on the response of the two major constituents, DPHA and DPPA. Samples taken from the WAFs prepared at loading rates of 10 and 18 mg/L were analyzed. The initial concentrations were 9.0 mg/L (DPPA) and 7.2 mg/L (DPHA) at the 10 mg/L WAF and 16 mg/L (DPPA) and 11 mg/L (DPHA) at the 18 mg/L WAF. These concentrations remained stable during the test period (102-105% of initial for DPPA and 87-94% of initial for DPHA at the end of the test). The test substance induced no lethal effects in carp at a loading rate of 10 mg/L (LL0) (equivalent to 7.2 mg/L of the constituent DPHA (LC0)). Overall, the deviation between the nominal loading levels and measured concentrations was less than 20%. Given these results, the effect parameters could be based on initial measured concentrations or even based on the initial loading rates (WAF). In absence of information on the actual moiety driving the toxicity, it was decided to base the effect concentrations on the initial loading rates. Based on nominal loading rate, the 96 h LL50 was 13 mg/L (equivalent to the measured concentration of 8.9 mg/L) (Bouwman, 2013).