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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:
12 Apr 2019 to 30 Apr 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals
Version / remarks:
OECD series on testing and assessment number 23, 2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: Pale yellow crystalline powder
Purity/Composition: 99.32%
Test item storage: At room temperature protected from light
Analytical monitoring:
yes
Details on sampling:
During the full test, samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 2).

Frequency 1. At the start of the test and after 72 hours from the freshly prepared solutions.
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
Volume 2.0 mL from the approximate centre of the test vessels
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. If not already used, these samples were stored in a freezer (≤-15°C) 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:
The batch of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide tested was a pale yellow crystalline powder with a purity of 99.32% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Test solutions were prepared under dimmed light conditions to minimize exposure to light.

Preparation of test solutions started with a loading rate of 100 mg/L applying an overnight period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by fitration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
Source: ‘De Haar Vissen’, Zodiac proefacc. Wageningen University and Research Centre, The Netherlands.
Mean length: 2.8 ± 0.3 cm
Mean weight(1): 0.24 ± 0.10 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: 42

(1) Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

Quarantine/Acclimatisation: At least 12 days after delivery.

Test medium: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
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 was continuously recorded. 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 0.5-0.8 mm, Alltech Coppens International B.V., 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:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
The hardness of test medium expressed as CaCO3: 180 mg/L
Test temperature:
20-24°C, constant within 2°C
pH:
6.0-8.5, constant within 1 unit
Dissolved oxygen:
oxygen > 60% of air saturation).
Salinity:
Not applicable
Nominal and measured concentrations:
The measured concentrations at the start of the test were 0.22, 0.51, 1.1, 2.3 and 5.0 mg/L in the fresh test solutions containing 4.6, 10, 22, 46 and 100% of the saturated solution
Details on test conditions:
Test duration : 96 hours
Test type: Semi-static, with daily renewal of test solutions
Test vessels: 6 litres, all-glass, containing 5 litres of test solution

Number of fish: 7 fish per test group
Loading: 0.34 g fish/Litre, i.e. 7 fish per 5 litres of test medium
Illumination: The test was performed under dimmed light conditions due to light sensitivity of the test item.
Aeration: The test solutions were not aerated during the test.
Feeding: No feeding from 24 hours prior to the test and during the total exposure period.
Introduction of fish: Within 0.75 hours 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 1.0 unit and 1.0°C.

Measurements and Recordings:
Mortality and other effects: At 2.75, 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 Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence interval between 0.93 and 2.1 mg/L
Reported statistics and error estimates:
The study met the acceptability criteria prescribed by the study plan and was considered valid.

Measured Test Item Concentrations

The results of analysis of the samples taken during the test are described in Table 2 of the appended Analytical Report.

Samples taken from the undiluted SS, all test concentrations and the control were analysed. The measured concentration in the freshly prepared and undiluted SS prepared at a loading rate of 100 mg/L was 5.0 mg/L at the start of the test and upon renewal after 72 hours of exposure. These results confirmed reproducibility of the preparation procedure. Small test item concentrations were detected in the analysed control samples. Since the fish exposed to the control treatment did not show any mortality, this was considered negligible and of no impact on the validity of the study interpretation.

The measured concentrations at the start of the test were 0.22, 0.51, 1.1, 2.3 and 5.0 mg/L in the fresh test solutions containing 4.6, 10, 22, 46 and 100% of the SS, respectively. Comparable values were measured in the freshly prepared solutions in the last refreshment period. During the first refreshment, concentrations remained stable, i.e. were in the range of 96 to 115% relative to the initial at the end of the 24 hour period. During both renewal periods, the concentrations remained stable, i.e. were at 87-98% of the initial concentrations in the 24 hour old test solutions.

Based on these results, the average exposure concentrations were calculated (see Table 1).

Table 1

Determination of Average Exposure Concentrations

Test Item1%SS prep. at 100 mg/L Measured concentration (mg/L) Average exposure conc. (mg/L)
t=0 h (fresh) t=24 h (old) t=72 h (fresh) t=96 h (old)
4.6 0.223 0.195 0.247 0.23 0.22
10 0.508 0.455 0.52 0.509 0.50
22 1.06 1.00 1.14 1.12 1.1
46 2.28 2.19 n.d. n.d. 2.2
100 4.96 4.86 * n.d. 4.9

1Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide;

n.d.: not determined, no surviving fish were present at this concentration;

* No surviving fish present. The concentration measured in the undiluted SS, that was used for preparing the lower test concentrations, was 5.03 mg/L.

Mortality and Clinical Effects

Table 2 shows the mortality data recorded during the test. Table 3 specifies the clinical effects observed at different test concentrations.

No mortality was observed in the control and at the two lowest test concentrations throughout the exposure period. After 72 hours of exposure, two fish exposed to an average concentration of 1.1 mg/L were observed to have lost their equilibrium as they were swimming sideways. After 96 hours of exposure, one fish showed this behaviour at this concentration, while another fish was found dead. All fish exposed to 2.2 mg/L were immobile after 24 hours of exposure and found dead after 48 hours of exposure. All fish exposed to 4.9 mg/L were found dead after 2.75 hours of exposure.

In addition, one fish in the control appeared to have lost its equilibrium as it was swimming sideways at the surface after 96 hours of exposure but survived throughout the test.

The responses recorded in this test allowed for reliable determination of an LC50.

Table 2

Incidence of Mortality and Total Mortality in the Full Test

Test Item1 Average conc. (mg/L) Initial number of fish Cumulative mortality Total Mortality (%)
2.75h 24h 48h 72h 96h
Control 7 0 0 0 0 0 0
0.22 7 0 0 0 0 0 0
0.50 7 0 0 0 0 0 0
1.1 7 0 0 0 0 1 14
2.2 7 0 0 7 7 7 100
4.9 7 7 7 7 7 7 100

1Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

Table 3

Clinical Effects Observed in the Full Test

Test Item1Average conc. (mg/L) Time of recording (hours) Specification of effects Relative number
Control 96 Swimming sideways at surface of test solution 01-Jul
1.1 72 Swimming sideways or upside down 02-Jul
96 Swimming sideways at surface of test solution 01-Jun
2.2 24 Immobile 07-Jul

1Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

Determination of Effect Concentrations

Table 4 shows the effect parameters based on average measured concentrations, see also Appendix 1.

Table 4

Effect Parameters

Parameter Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide Average conc. (mg/L) 95%-confidence interval
24h-LC50 3.3 2.2-4.9
48h, 72h-LC50 1.6 1.1-2.2
96h-LC50 1.4 0.93-2.1

Experimental Conditions

The results of measurement of pH and oxygen concentrations are presented in Table 5 and Table 6. The temperatures measured during the study in the various test vessels are presented in Table 7.

All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Table 5

pH-Values During the Full Test

Test Item1Average conc. (mg/L) Day 02 Day 1 Day 2 Day 3 Day 4
Fresh Old Fresh Old Fresh Old Fresh Old
Control 7.6 7.6 7.5 7.8 7.8 7.8 7.6 7.7
0.22 7.6 7.5 7.5 7.7 7.6 7.6 7.6 7.7
0.5 7.6 7.5 7.4 7.7 7.6 7.6 7.6 7.7
1.1 7.6 7.5 7.5 7.7 7.6 7.6 7.6 7.7
2.2 7.6 7.5 7.5 7.7 n.d. n.d. n.d. n.d.
4.9 7.5 n.d. n.d. n.d. n.d. n.d. n.d.

n.d.

1Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide; 2 pH of culture medium was: 8.0;

n.d.: not determined, no surviving fish were present at this concentration.

Table 6

Dissolved Oxygen Concentrations (mg/L) During the Full Test

Test Item1 Day 0 Day 1 Day 2 Day 3 Day 4
Average conc. (mg/L) Fresh Old Fresh Old Fresh Old Fresh Old
Control 9.1 7.6 8.7 7.9 8.9 8.1 9.2 8.4
0.22 9.1 7 8.6 7.2 8.9 7.5 9.2 7.9
0.5 9.1 7.2 8.5 7.7 8.9 7.9 9.2 8.2
1.1 9.1 7.5 8.5 8.0 8.9 8.1 9.1 8.5
2.2 9.0 7.4 8.3 8.4 n.d. n.d. n.d. n.d.
4.9 8.8 n.d. n.d. n.d. n.d. n.d. n.d. n.d.

1Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide;

n.d.: not determined, no surviving fish were present at this concentration.

Table 7

Temperatures (°C) Measured During the Full Test

Test Item1Average conc. (mg/L) Day 02 Day 1 Day 2 Day 3 Day 4
Fresh Old Fresh Old Fresh Old Fresh Old
Control 21 21 20 21 21 21 21 21
0.22 21 21 21 21 21 21 21 21
0.50 21 21 21 21 21 21 21 21
1.1 21 21 21 21 21 21 21 21
2.2 21 21 21 21 n.d. n.d. n.d. n.d.
4.9 21 n.d. n.d. n.d. n.d. n.d. n.d. n.d.

1Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide; 2 Temperature of culture medium was: 21°C;

n.d.: not determined, no surviving fish were present at this concentration.

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the 96h-LC50 was 1.4 mg/L based on average exposure concentrations (95% confidence interval between 0.93 and 2.1 mg/L).
Executive summary:

The objective of the study was to evaluate Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide for its ability to generate acute toxic effects in Cyprinus carpio during an exposure period of 96 hours and, if possible, to determine the LC50 at all observation times.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2019.

The batch of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide tested was a pale yellow crystalline powder with a purity of 99.32% and not completely soluble in test medium at the loading rate initially prepared.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. Test solutions were prepared under dimmed light conditions to minimize exposure to light.

A full test was performed. Seven fish per group were exposed to an untreated control and to solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L. Since the test item was light sensitive, exposure was performed under dimmed light conditions. The total exposure period was 96 hours. Test solutions were renewed every 24 hours of exposure and samples for analytical confirmation of actual exposure concentrations were taken during two renewal intervals from freshly prepared and 24-hour old solutions.

Samples taken from all test concentrations and the control were analysed. Based on these results, the average exposure concentrations were calculated to be 0.22, 0.50, 1.1, 2.2 and 4.9 mg/L in solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L.

No mortality was observed in the control and at the two lowest test concentrations throughout the exposure period. After 72 hours of exposure, two fish exposed to an average concentration of 1.1 mg/L were observed to have lost their equilibrium as they were swimming sideways. After 96 hours of exposure, one fish showed this behaviour at this concentration, while another fish was found dead. All fish exposed to 2.2 mg/L were immobile after 24 hours of exposure and found dead after 48 hours of exposure. All fish exposed to 4.9 mg/L were found dead after 2.75 hours of exposure.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 96h-LC50 was 1.4 mg/L based on average exposure concentrations (95% confidence interval between 0.93 and 2.1 mg/L).

Description of key information

Study conducted to recognised testing guideline with GLP certification.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.4 mg/L

Additional information