4-hydroxybenzophenone

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted April 13, 2004

Deviations:

no

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 all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis. The method of analysis is described in the appended Analytical Report (Appendix 3).

Frequency at t=0 h and t=48 h
Volume 3.0 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.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 3.0 mL were taken for possible analysis. If not 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 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 combined limit/range-finding test, all test solutions were clear and colourless at the end of the preparation procedure. In the final test, all test concentrations were observed to be clear and colourless, except the highest test concentration which was observed to be clear and lightly yellow.

Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%1, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding: Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.

Maximum age of the cultures: 4 weeks.

Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.

Feeding: Daily, a suspension of fresh water algae.

Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

The following salts and vitamins were added to freshly prepared test medium to reach the following concentrations:

Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins:
Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

18-22°C

pH:

6-9

Dissolved oxygen:

8.7 mg/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

The measured concentrations were 9.7, 18, 32, 55 and 99 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 97-98% relative to the initial concentration levels.

Details on test conditions:

The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a SS prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:

- Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to solutions containing 1.0 and 10% of the SS prepared at a loading rate of 100 mg/L in the combined range-finding test.

- Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Test duration: 48 hours.

Test type: Static.

Test vessels: 60 mL, all-glass.

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

Number of daphnids: 20 per concentration.

Loading: 5 per vessel containing 50 mL of test solution.

Light: A daily photoperiod of 16 hours.

Feeding: No feeding.

Aeration: No aeration of the test solutions was applied.

Introduction of daphnids: 14 minutes after preparation of the test solutions.

Reference substance (positive control):

no

Remarks:

Reference substance was negative control: test medium without test item or other additives

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% confidence interval between 11 and 15 mg/L

Details on results:

Combined Limit/Range-Finding Test:
Table 1 shows the responses recorded during the combined limit/range-finding test.

No immobility was observed in the control and the two lowest test concentrations throughout the test, except for a single daphnid in the control at the end of the test. At the highest test concentration, all daphnids were observed to be immobile from 24 hours of exposure onwards.

Based on these results samples taken from solutions containing 10 and 100% of the SS were analysed. The initial concentrations were 9.6 and 98 mg/L, respectively. These concentration remained stable during the test period, i.e. were at 99-100% 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.

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 9.7, 18, 32, 55 and 99 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 97-98% 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. Considering these results, the effect parameters were based on analytically confirmed nominal concentrations.

Immobility:
Table 2 shows the responses recorded during the final test.

No immobility was observed in the control throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at all test concentrations reaching complete immobilization at the three highest test concentrations.

The responses recorded in this test allowed for reliable determination of an EC50.

Determination of Effect Concentrations:
Table 3 shows the effect parameters based on analytically confirmed nominal concentrations, see also Appendix 1.

Experimental Conditions:
The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 4. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: 3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within ± 1°C).

Results with reference substance (positive control):

See Appendix 2, "Reference Test", attached below.

Reported statistics and error estimates:

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

Table 1

Number of Introduced Daphnids and Incidence of Immobility in the Combined Limit/Range-Finding Test

		4-Hydroxy-benzophenone; %SS prep. at 100 mg/L
Time (h)	Replicate	Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	5^#
	B	0	0	0	5
	C	0			5
	D	0 (1)			5
	Total immobilised	0	0	0	20
	Effect %	0	0	0	100
48	A	0	0	0	5
	B	1^*	0	0	5
	C	0			5
	D	0			5
	Total immobilised	0	0	0	20
	Effect %	0	0	0	100

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

^#Microscopic observation revealed no test item attached to the daphnids.

^*Found above the surface of the test solution.

Table 2

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

		4-Hydroxy-benzophenone; Nominal conc. (mg/L)
Time (h)	Replicate	Control	10	18	32	56	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	1	2	5^*	5	5
	B	0	0	3	5	5	5
	C	0	0	1	5	5	5
	D	0	0	4^#	5	5	5
	Total immobilised	0	1	10	20	20	20
	Effect %	0	5	50	100	100	100
48	A	0	1	4	5	5	5
	B	0	1	5	5	5	5
	C	0	0	5	5	5	5
	D	0	0	5	5	5	5
	Total immobilised	0	2	19	20	20	20
	Effect %	0	10	95	100	100	100

^#Microscopic observation revealed no test item attached to the daphnids.

^*Dissolved oxygen: 8.7 mg/L

Table 3

Effect Parameters

Parameter	4-Hydroxy-benzophenone Nominal conc. (mg/L)	95%-confidence interval (mg/L)
24h-EC50	18	15-20
48h-EC50	13	11-15

Table 4

pH and Oxygen Concentrations (mg/L) During the Final Test

	Start (t=0 h)		End (t=48 h)
4-Hydroxy-benzophenone Nominal conc. (mg/L)	O2	pH	O2	pH
Control	8.5	8.0	8.8	8.1
10	8.9	7.8	8.6	8.0
18	8.9	7.7	8.5	8.0
32	8.9	7.6	8.5	8.0
56	8.9	7.5	8.5	7.9
100	8.6	7.4	8.5	7.8

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to 4-Hydroxy-benzophenone was 13 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 11 and 15 mg/L).

Executive summary:

The objective of the study was to evaluate 4-Hydroxy-benzophenone for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC₅₀ at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. 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 combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) 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 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from the control and all test concentrations were analysed. The measured concentrations were 9.7, 18, 32, 55 and 99 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 97-98% relative to the initial concentration levels. Considering these results, the effect parameters were based on analytically confirmed nominal concentrations.

No immobility was observed in the control throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at all test concentrations reaching complete immobilization at the three highest test concentrations.

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

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to 4-Hydroxy-benzophenone was 13 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 11 and 15 mg/L).

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

13 mg/L