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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 16 July, 2012 to 11 Aug, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
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:
no
Qualifier:
according to
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
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations with surviving fish and the control according to the schedule below. In addition, a sample was taken from the highest test concentration after 19 h of exposure when all fish were found dead.
Frequency: At t=0 h, t=24 h and t=96 h
Volume: 1.5 mL
Storage: Samples were stored in a freezer until analysis.

Additionally, reserve samples of 1.5 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:
yes
Details on test solutions:
Ebecryl® 140 radiation curing resins, 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 h where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned off and used as test concentrations.
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: 3.2 ± 0.4 cm
Final test: 3.0 ± 0.1 cm

Mean weight:
Range-finding test: 1.00 ± 0.45 g
Final test: 0.79 ± 0.09 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.
Quarantine/Acclimatisation: At least 12 d after delivery.
Feeding: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the 7 d 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:
hardness of 180 mg CaCO3 per litre
Test temperature:
Between 21.7 and 22.2 °C
pH:
Between 7.3 and 8.3
Dissolved oxygen:
Between 6.2 and 8.8 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Measured concentration: Analysis of the samples taken at the start of the final test showed measured concentrations of 1.1, 2.1 and 3.2 mg/L at the WAFs prepared at loading rates of 3.2, 5.6 and 10 mg/L. The highest test concentration remained stable during 19 h of exposure (91% of initial). The two lower concentrations remained relatively stable during 24 h of exposure (77-88% of initial), where after the lowest test concentration decreased to 53% of initial at the end of the test. Based on these results, the average exposure concentrations were calculated to be 0.81, 1.9 and 3.1 mg/L.
Details on test conditions:
Test duration: 96 h
Test type: Static
Test vessels: 20 L, all-glass, containing 15 L 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
Loading: 0.37 g fish/litre, i.e. 7 fish per 15 L of test medium
Illumination: 16 h photoperiod (dimmed light) daily
Aeration: The test media were not aerated during the test
Feeding: No feeding from 24 h prior to the test and during the total test period
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.
Reference substance (positive control):
yes
Remarks:
pentachlorophenol (PCP)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% C.I. 0.81-1.9 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
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):
The 96h-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.
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.

Range-finding test:

After 24 h of exposure all fish exposed to the WAFs prepared at loading rates of 10 and 100 mg/L were dead. No mortality or clinical effects were observed at the lower loading rate of 1.0 mg/L during the total test period. Based on these results, samples taken from the WAFs prepared at loading rates of 1.0 and 10 mg/L were analysed. The initial concentrations were 0.68 and 4.5 mg/L, respectively. These concentrations decreased to 66-67% of initial after 24 h of exposure and further to 50-61% of initial at the end of the test. The expected LC50 was between time weighted average (TWA) concentrations of 0.43 and 3.0 mg/L. All test conditions were maintained within the limits prescribed by the protocol.

 

Final test

-Measured concentrations: Analysis of the samples taken at the start of the final test showed measured concentrations of 1.1, 2.1and 3.2 mg/L at the WAFs prepared at loading rates of 3.2, 5.6 and 10 mg/L. The highest test concentration remained stable during 19 h of exposure (91% of initial). The two lower concentrations remained relatively stable during 24 h of exposure (77-88% of initial), where afterthe lowest test concentration decreased to 53% of initial at the end of the test. Based on these results, the average exposure concentrations were calculated to be 0.81, 1.9 and 3.1 mg/L.

-Mortality and other effects: All fish exposed to the two highest test concentrations died during the test period, while all fish exposed to the lower test concentrations andthe control survived. The responses recorded in this test allowed for reliable determination of an LC50. No clinical effects were observed, except for the observation of a precipitate in the test medium of the two highest test concentrations after 24 h of exposure. This precipitate looked like fish scales butit was not clear if fish lost their scales before or after death.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the NOEC for di-TMPTTA in carp was 0.81 mg/L. The 96 h LC50 was 1.2 mg/L (95% confidence interval between 0.81 and 1.9 mg/L) (WAF).
Executive summary:

A study was conducted to assess the acute toxicity of di-TMPTTA to common carp (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) of the test substance prepared at loading rates of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The total test period was 96 h and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 h of exposure and at the end of the test. In addition, a sample was taken from the highest test concentration after 19 h of exposure when all fish were found dead. Analysis of the samples taken at the start of the final test showed measured concentrations of 1.1, 2.1 and 3.2 mg/L at the WAFs prepared at loading rates of 3.2, 5.6 and 10 mg/L. The highest test concentration remained stable during 19 h (91% of initial). The two lower concentrations remained relatively stable during 24 h (77-88% of initial), after which the lowest test concentration decreased to 53% of initial at the end of the test. Based on these results, the average exposure concentrations were calculated to be 0.81, 1.9 and 3.1 mg/L. Under the test conditions, the test substance induced no visible effects in carp at or below 0.81 mg/L (NOEC). The 96 h LC50 was 1.2 mg/L (95% confidence interval between 0.81 and 1.9 mg/L) and was already reached after 24 h of exposure (Bouwman, 2013).

Description of key information

Based on the study results, the NOEC for the test substance in carp was 0.81 mg/L. The 96 h LC50 was 1.2 mg/L (95% confidence interval between 0.81 and 1.9 mg/L) (measured; WAF).

Key value for chemical safety assessment

LC50 for freshwater fish:
1.2 mg/L

Additional information

A study was conducted to assess the acute toxicity of the test substance to common carp (Cyprinus carpio) in accordance with OECD Guideline 203, EU Method C.1 and ISO International Standard 7346-1. Seven fish per concentration were exposed to water accommodated fractions (WAFs) of the test substance prepared at loading rates of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The total test period was 96 h and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 h of exposure and at the end of the test. In addition, a sample was taken from the highest test concentration after 19 h of exposure when all fish were found dead. Analysis of the samples taken at the start of the final test showed measured concentrations of 1.1, 2.1 and 3.2 mg/L at the WAFs prepared at loading rates of 3.2, 5.6 and 10 mg/L. The highest test concentration remained stable during 19 h (91% of initial). The two lower concentrations remained relatively stable during 24 h (77-88% of initial), after which the lowest test concentration decreased to 53% of initial at the end of the test. Based on these results, the average exposure concentrations were calculated to be 0.81, 1.9 and 3.1 mg/L. Under the test conditions, the test substance induced no visible effects in carp at or below 0.81 mg/L (NOEC). The 96 h LC50 was 1.2 mg/L (95% confidence interval between 0.81 and 1.9 mg/L) and was already reached after 24 h of exposure.