4-hydroxybenzophenone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

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:

Frequency at t=0 h and t=72 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 with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10% of the SS but without algae and samples for analysis were taken at the start and at the end of the test period.

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 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, except the highest test concentration which was observed to be clear and lightly yellow. In the final test, all test concentrations were observed to be clear and colourless.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Any residual volumes were discarded.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:

NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 °C

pH:

8.1 ± 0.2

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Solutions containing 1.0, 3.2, 10, 10*, 32 and 100% of the SS prepared at a loading rate of 100 mg/L.
*Without algae

At t=0 h: 1.0, 3.2, 10, 10*, 32 and 100% of the SS prepared at a loading rate of 100 mg/L
At t=72 h: 1.0, 3.2, 10, 10*, 32 and 100% of the SS prepared at a loading rate of 100 mg/L
*Without algae

Details on test conditions:

The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae 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:

Three replicates per concentration 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.

Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period. Cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm).

pH was only measured in the control and the highest test concentration.

At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

Samples for possible analysis were additionally taken after 24 hours of exposure. To this end, one extra replicate of each test group was included for sampling purposes.

Cell density An initial cell density of 1E4 cells/mL.

Illumination Continuously using TLD-lamps with a light intensity within the range of 88 to 92 µE.m^-2.s^-1.

Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings:
pH: At the beginning and at the end of the test, for all test concentrations and the control.

Temperature of medium: Continuously in a temperature control vessel.

Appearance of the cells: At the end of the final test, microscopic observations were performed on the 3.2 and 10% SS test concentrations and the control to observe for any abnormal appearance of the algae.

Recording of Cell Densities:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

statistical significance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Remarks:

Statistical significance and biological relevance

Details on results:

Combined Limit/Range-Finding Test:
The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

A concentration-related inhibition of algal growth rate and yield was found at all test concentrations, resulting in complete inhibition of algal growth at the highest test concentration at the end of the test.

Based on these results, samples taken from solutions containing 1.0 and 100% of the SS prepared at a loading rate of 100 mg/L were analysed. The measured concentrations at the start of the test were 0.96 and 94 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 94-103% 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.

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 all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.80. 2.5, 7.9, 26 and 79 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 90-103% relative to the initial concentrations at the end of the test.

The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae, indicating that the presence of the algae did not affected the concentration of the test item in test medium throughout the test.

It should be noted that a small concentration was measured in the control at the end of the test. Considering that no concentration was measured in the sample taken at the start of the test and that the validity criteria were met, it was assumed that the sample was contaminated during the course of sampling at the end of the test.

Based on these results, the measured exposure concentrations were used to determine the effect parameters (see Appendix 4).

Mean Cell Densities:
Figure 1 shows growth curves at different concentrations of 4-Hydroxy-benzophenone. The individual and group mean cell densities measured at 24h intervals are given in Table 9.

Inhibition of Growth Rate and Inhibition of Yield:
Table 4 shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereas Table 5 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 6 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 2 and Appendix 3.

A concentration-related inhibition of algal growth rate and yield was found at all test concentrations, resulting in 92% and 100% inhibition of algal growth rate and yield, respectively, at the highest test concentration at the end of the test.

Statistically significant inhibition of growth rates was found at test concentrations of 2.5 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 2.5 mg/L, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 2.5 mg/L. Statistically significant inhibition of yield was found at test concentrations of 2.5 mg/L and higher.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 2.5 and 7.9 mg/L when compared to the control.

Determination of Effect Concentrations:
Table 7 shows the effect parameters based on measured concentrations, see also Appendix 4.

Experimental Conditions:
Table 8 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained at 22°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within ±1°C).

Table 1

Mean Cell Densities (x10^4 Cells/mL) during the Combined Limit/Range-Finding Test

	4-Hydroxy-benzophenone; %SS prep. at a loading rate of 100 mg/L
Time (h)	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	4.9	n.d.	n.d.	1.3
48	34	n.d.	n.d.	3.0
72	176	144	13	1.0

Table 2

Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

4-Hydroxy-benzophenone %SS prep. at a loading rate of 100 mg/L	Mean	Std. Dev.	n	%inhibition
Control	1.722	0.0136	6
1.0	1.658	0.0087	3	3.8
10	0.863	0.0289	3	50
100	0.000	0.0000	6	100

Table 3

Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

4-Hydroxy-benzophenone %SS prep. at a loading rate of 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	174.598	7.2563	6
1.0	143.455	3.7890	3	18
10	12.346	1.1370	3	93
100	0.0000	0.0000	6	100

Table 4

Growth Rate and Percentage Inhibition for the Total Test Period

4-Hydroxy-benzophenone Measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.781	0.0145	6
0.80	1.764	0.0177	3	0.93
2.5	1.679	0.0112	3	5.7^#
7.9	1.173	0.0373	3	34^*
26	0.2	0.0636	3	89^*
79	0.147	0.0508	3	92^*

^* Effect was statistically significant.; ^# Effect was statistically significant, but biologically not relevant (<10%).

Table 5

Growth Rate and Percentage Inhibition at Different Time Intervals

4-Hydroxy-benzophenone Measured conc. (mg/L)	n	Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.186		1.715		1.443
0.80	3	2.215	-1.4	1.674	2.4	1.404	2.7
2.5	3	2.224	-1.8	1.520	11	1.292	10
7.9	3	2.099	4.0	0.731	57	0.688	52
26	3	0.962	56	0.332	81	-0.693	148
79	3	0.559	74	-0.402	123	0.284	80

Table 6

Yield and Percentage Inhibition for the Total Test Period

4-Hydroxy-benzophenone Measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	208.311	9.0414	6
0.80	198.193	10.7000	3	4.9
2.5	153.008	5.1661	3	27^*
7.9	32.849	3.8425	3	84^*
26	0.847	0.3576	3	100^*
79	0.566	0.2442	3	100^*

^*Effect was statistically significant.

Table 7

Effect Parameters

Parameter (mg/L)		NOEC^*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.80	2.5	3.8	5.4	11
	lower 95%-cl			3.5	5.1	10
	upper 95%-cl			4.1	5.7	11
Yield	Value	0.80	0.80	1.5	2.1	3.9
	lower 95%-cl			1.4	2.0	3.7
	upper 95%-cl			1.6	2.2	4.0

cl: confidence limit; ^*based on statistical significance; ^#based on biological relevance.

Table 8

pH Levels Recorded during the Final Test

	pH
4-Hydroxy-benzophenone Measured conc. (mg/L)	t=0h	t=72h
Control	8.1	8.0
0.80	8.1	8.0
2.5	8.1	7.8
7.9	8.0	7.8
26	7.9	7.8
79	7.7	7.7

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, 4-Hydroxy-benzophenone inhibited growth rate and yield of this fresh water algae species significantly at measured concentrations of 2.5 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 11 mg/L with a 95% confidence interval ranging from 10 to 11 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 3.9 mg/L with a 95% confidence interval ranging from 3.7 to 4.0 mg/L.

The 72h-NOEC for growth rate inhibition was 0.80 mg/L based on statistical significance and 2.5 mg/L based on biological relevance.

The 72h-NOEC for yield inhibition was 0.80 mg/L based on statistical significance and biological relevance.

Executive summary:

The objective of the study was to evaluate 4-Hydroxy-benzophenone for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the testmethods 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. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.80. 2.5, 7.9, 26 and 79 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 92-103% relative to the initial concentrations at the end of the test. Based on these results, the measured exposure concentrations were used to determine the effect parameters.

A concentration-related inhibition of algal growth rate and yield was found at all test concentrations, resulting in 92% and 100% inhibition of algal growth rate and yield, respectively, at the highest test concentration at the end of the test.

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

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC^*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.80	2.5	3.8	5.4	11
	lower 95%-cl			3.5	5.1	10
	upper 95%-cl			4.1	5.7	11
Yield	Value	0.80	0.80	1.5	2.1	3.9
	lower 95%-cl			1.4	2.0	3.7
	upper 95%-cl			1.6	2.2	4.0

cl: confidence limit; ^*based on statistical significance; ^#based on biological relevance.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, 4-Hydroxy-benzophenone inhibited growth rate and yield of this fresh water algae species significantly at measured concentrations of 2.5 mg/L and higher.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L

EC10 or NOEC for freshwater algae:

0.8 mg/L