4-hydroxybenzophenone

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Dec 2018 to 23 Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

Temperature differed by 2ºC between two highest test solutions & holding tank. However the temperature difference did not affect the integrity of the study or the interpretation of the test results as toxicity was observed at lower concentrations.

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals

Version / remarks:

OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: White powder
Purity/Composition: 99.72%, assumed 100% for testing
Test item storage: At room temperature desiccated

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept forpossible analysis. In cases of treatments where all fish died before the end of exposure, the samples were taken directly after the last fish was found dead. The method of analysis is described in the appended Analytical Report (Appendix 2).

Frequency: at t=0 h, t=24 h and t=96 h
Volume: 3.0 mL
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for possible analysis 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 4-Hydroxy-benzophenone tested was a white powder with a purity of 99.72% and visually not completely soluble in test medium at the loading rate originally 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 support maximum dissolution of the test item in test medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration 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. In the range-finding test, all test solutions were clear and colourless at the end of the preparation procedure. In the final test, the lowest test concentration was observed to be clear and colourless, while the higher test concentrations were observed to be clear and very light to light yellow.

Any residual volumes were discarded.

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(1): Range-finding test: 3.5 ± 0.4 cm, Final test: 2.5 ± 0.1 cm

Mean weight(1) Range-finding test: 0.48 ± 0.20 g, Final test: 0.19 ± 0.1 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

Holding:
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

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

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:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

180 mg/L CaCO3

Test temperature:

20-24°C, constant within 2°C

pH:

7.7 ± 0.3

Dissolved oxygen:

7.2 - 9.5 mg/L

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

The measured concentrations were 11, 18, 33, 57 and 102 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L

Details on test conditions:

Range-Finding Test
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three fish per concentration were exposed to solutions containing 1.0 to 100% of a SS prepared at a loading rate of 100 mg/L increasing by a factor of 10.
- Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration with surviving fish.

Final Test
Test Concentrations
4-Hydroxy-benzophenone: Solutions containing 10, 18, 32, 56 and 100% of a SS prepared at a loading rate of 100 mg/L.
Controls: Test medium without test item or other additives.

Test Procedure and Conditions
Test duration: 96 hours
Test type: Static
Test vessels: 6.5 litres, all-glass, containing 5.0 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
Loading: 0.27 g fish/Litre, i.e. 7 fish per 5 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 36 minutes after preparation of the test solutions from a holding tank with comparable water quality parameters and pH
and temperature differences between test and holding media of less than 1 unit and 1°C, except for the highest test concentration where the temperature differed by 2°C from the holding tank 2.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol - details in Appendix 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

95% confidence interval between 10 and 18 mg/L

Results with reference substance (positive control):

See Appendix 1

Reported statistics and error estimates:

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

Sublethal observations / clinical signs:

Range-Finding test

The results of the range-finding test are presented in Table 1. No mortality or clinical effects were observed at the two lowest test concentrations during the test period, while at the highest test concentrations all fish were dead after 24 hours of exposure.

Based on these results samples taken from solutions containing 10 and 100% of the SS were analysed. The initial concentrations were 10 and 100 mg/L, respectively. These concentrations remained stable during the test period, i.e. were at 94-100% relative to the initial concentrations at the end of the test (see also Table 2 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Table 1

Incidence of Mortality and Total Mortality During the Range-Finding Test

4-Hydroxy-benzophenone % SS prep. at 100 mg/L	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2.25h	24h	48h	72h	96h
1.0	3	0	0	0	0	0	0
10	3	0	0	0	0	0	0
100	3	0	3	3	3	3	100

Final Test

Measured Test Item Concentrations

The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report. Samples taken from the control and all test concentrations were analysed. The measured concentrations were 11, 18, 33, 57 and 102 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L. These concentrations remained stable during the test period, i.e. were at 97-100% relative to the initial concentration levels. Based on these results, it was concluded that that the undissolved fraction removed during the preparation of the test solutions was not test item related.

It should be noted that a small response was measured in the control at the start of the test. Considering the test item stability, that no concentration was measured at the end of the test, and that no effects were observed in the control group of fish, it was assumed that the sample had likely been contaminated during the sampling process.

Considering these results, the effect parameters were based on analytically confirmed nominal concentrations.

Mortality and Other Effects

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

No mortality was observed in the control and at the lowest test concentration throughout the test. From 48 hours of exposure onwards, six of the surviving fish at the lowest test concentration were observed to be gasping at the surface of the test solution. None of the fish exposed to 18 mg/L survived the first 24 hours of exposure. While at the higher concentrations, none of the fish survived the first 2.25 hours of exposure. The responses recorded in this test allowed for reliable determination of LC₅₀-values.

Table 2

Incidence of Mortality and Total Mortality During the Final Test

4-Hydroxy-benzophenone Nominal conc. (mg/L)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2.25h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
10	7	0	0	0	0	0	0
18	7	0	7	7	7	7	100
32	7	7	7	7	7	7	100
56	7	7	7	7	7	7	100
100	7	7	7	7	7	7	100

Table 3

Clinical Effects Observed During the Final Test

4-Hydroxy-benzophenone Nominal conc. (mg/L)	Time of recording (hours)	Specification of effects	Relative number of affected fish
10	48-96	Gasping at the surface of the test solution	6/7
18	2.25	Gasping at the surface of the test solution Loss of equilibrium	7/7 2/7

Determination of Effect Concentrations

Table 4 shows the effect parameters based on analytically confirmed nominal concentrations.

Table 4

Effect Parameters

Parameter	4-Hydroxy-benzophenone Nominal conc. (mg/L)	95%-confidence interval (mg/L)
24h, 48h, 72h, 96h -LC50	13	10-18

Experimental Conditions

The results of measurement of pH and oxygen concentrations are presented in Table 5. The temperatures measured during the study in the various test vessels are presented in Table 6. 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 and Dissolved Oxygen Concentrations (mg/L) During the Final Test

4-Hydroxy-benzophenone Nominal conc. (mg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH^1	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control	7.8	9.0	8.0	8.3	7.9	8.2	7.9	7.7	8.1	7.8
10	7.8	9.1	7.9	7.8	7.7	7.9	7.7	7.3	7.9	7.2
18	7.8	9.2	7.9	9.1	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
32	7.7	9.2^2	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
56	7.7^2	9.3^2	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
100	7.7^2	9.5^2	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.

¹ pH of culture medium was: 8.1;

² No significant change when measured again after 2.25 hours of exposure;

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

Table 6

Temperatures (°C) Measured During the Final Test

4-Hydroxy-benzophenone Nominal conc. (mg/L)	Day 0^1	Day 1	Day 2	Day 3	Day 4
Control	21	21	20	21	21
10	21	21	20	21	21
18	20	21	n.d.	n.d.	n.d.
32	20^2	n.d.	n.d.	n.d.	n.d.
56	20^2	n.d.	n.d.	n.d.	n.d.
100	19^2	n.d.	n.d.	n.d.	n.d.

¹ Temperature of culture medium was: 21°C;

² No significant change when measured again after 2.25 hours of exposure;

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

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 96h-LC50 was 13 mg/L based on analytically confirmed nominal exposure concentrations (95% confidence interval between 10 and 18 mg/L) and was already reached after 24 hours of exposure.

Executive summary:

The objective of the study was to evaluate 4-Hydroxy-benzophenone 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, 2000.

The batch of 4-Hydroxy-benzophenone tested was a white powder with a purity of 99.72% and visually not completely soluble in test medium at the loading rate originally 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.

A final test was performed based on the results of a preceding range-finding test. Seven fish per group were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100% of a SS prepared at a loading rate of 100 mg/L. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and after 96 hours of exposure.

Samples taken from the control and all test concentrations were analysed. The measured concentrations were 11, 18, 33, 57 and 102 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L. These concentration remained stable during the test period, i.e. were at 97-100% relative to the initial concentration levels. Considering these results, the effect parameters were based on analytically confirmed nominal concentrations.

No mortality was observed in the control and at the lowest test concentration throughout the test. At 24 hours of exposure, all fish exposed to 18 mg/L were dead. At all higher test concentrations, all fish were dead after 2.25 hours of exposure.

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

In conclusion, the 96h-LC₅₀ was 13 mg/L based on analytically confirmed nominal exposure concentrations (95% confidence interval between 10 and 18 mg/L) and was already reached after 24 hours of exposure.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

13 mg/L