Boronic acid, (4-ethylphenyl)-

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:

2007-10-24 to 2008-02-26

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

December 29, 1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken at the beginning of the study and after 72 hours. The samples were delivered frozen to the analytic laboratory for quantitative analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For this purpose, the test material was weighed into freshly reconstituted water and then placed into an ultrasonic device for one hour. Then, the preparation was stirred for 23 hours. After stirring the preparation was passed through a single use syringe filter (pore size 0.2 µm). The filtrate was diluted with reconstituted water to the different test media concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen


ACCLIMATION
Prior to start the study, the algae were grown in reconstituted water as preculture and the growth rate was determined.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 - 24 °C

pH:

7.63 - 7.89 (without algae);
7.63 – 10.29 (with algae)

Nominal and measured concentrations:

Nominal conc.: 10.0, 17.8, 31.6, 56.2, and 100.0 mg/L;
Measured conc.: between 98.9 - 100.0 % (at the start) and 99.6 - 101.0 % of the nominal concentration (at the end of exposure)

Details on test conditions:

TEST SYSTEM
- Test vessel, size, fill volume: Erlenmeyer flasks, 300 mL, 100 mL
- Type: closed
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The algae cell densities were determined with an electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78

RANGE FINDING STUDY
- Test concentrations: 10.0 – 100.0 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 54.6 - 64.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence interval: 35.0 - 40.6 mg/L

Details on results:

In the preculture, the factor of cell growth was approx. 120.7. The algae increased from 10,000 cells/mL at the start to 1,206,695 cells/mL after 72 hours in the test material concentration group of 10 mg/L.

Results with reference substance (positive control):

Results not specified in the report

Algae growth

The nominal test material concentrations and the growth inhibition data after 72 hours exposure are given below:

 

Test material (mg/L)	Cell density per mL
0.0		0 hour	24 hours	48 hours	72 hours
		10,000	58,527	236,300	1,009,503
		10,000	64,800	227,069	1,063,530
		10,000	63,837	247,129	1,111,320
	mean	10,000	62,388	236,833	1,061,451
10.0		10,000	75,960	254,320	1,298,997
		10,000	79,650	274,720	1,350,027
		10,000	78,903	275,910	1,483,920
	mean	10,000	78,171	268,317	1,377,648
17.8		10,000	67,923	195,500	1,074,870
		10,000	68,643	202,759	1,059,723
		10,000	71,037	202,300	1,030,077
	mean	10,000	69,201	200,186	1,054,890
31.6		10,000	57,357	130,023	569,211
		10,000	58,797	137,520	651,321
		10,000	60,660	149,220	688,381
	mean	10,000	58,938	138,921	636,304
56.2		10,000	37,503	53,757	115,567
		10,000	37,557	52,443	108,207
		10,000	38,097	52,857	110,367
	mean	10,000	37,719	53,019	111,387
100.0		10,000	34,947	27,423	23,733
		10,000	30,780	23,760	19,683
		10,000	31,293	23,220	20,133
	mean	10,000	32,340	24,801	21,183

 

Test material (mg/L)	Inhibition of cell growth in %			Growth inhibition (0-72 hours)
	24 hours	48 hours	72 hours	IA	Iµt
10.0	-30.1	-16.9	-27.2	-25.5	-5.6
17.8	-13.0	10.7	3.4	4.1	0.1
31.6	6.6	36.3	40.9	39.0	11.0
56.2	47.1	74.7	88.7	84.9	48.3
100.0	57.4	86.7	98.0	94.7	83.9

 

I_A= inhibition of cell growth in percent

I_µt= inhibition of growth rate in percent

 

Temperature, pH value

Temperature and pH values were inconspicuous.

 

Concentration (mg/L)	pH without algae		pH with algae
	Start of the experiment	End of the experiment	Start of the experiment	End of the experiment
0.0	7.87	7.85	7.87	10.14
10.0	7.74	7.88	7.74	10.29
17.8	7.72	7.89	7.72	9.52
31.6	7.71	7.88	7.71	8.75
56.2	7.67	7.86	7.67	7.95
100.0	7.63	7.82	7.63	7.83

The temperature during the experimental part, measured in a control flask with an electronic thermometer containing a maximum and minimum memory display, ranged from 23 to 24 °C.

Analytical results

Results for determination of the test material in the test samples are given in the following table:

Group No.	Sample No.	Nominal concentration mg/L	Time after preparation [hours]	Mean analytical concentration [mg/L]	% of nominal concentration
1	1	100.0	0 (start)	997	99.7
	6		72 (end)	100.3	100.3
2	2	56.2	0 (start)	55.8	99.3
	7		72 (end)	56.0	99.6
3	3	31.6	0 (start)	31.5	99.7
	6		72 (end)	31.6	100.0
4	4	17.8	0 (start)	17.6	98.9
	9		72 (end)	17.9	100.6
5	5	10.0	0 (start)	10.0	100.0
	10		72 (end)	10.1	101.0

Validity criteria fulfilled:

yes

Conclusions:

Under the given conditions of this study, the 72-h ErC50 value was determined to be 59.3 mg/L (95 % CI: 54.6 - 64.4 mg/L). The 72-hour NOEC was determined to be 17.8 mg/L.

Executive summary:

The acute toxicity of the test item was investigated in a GLP-study according to OECD TG 201 using unicellular freshwater green alga Desmodesmus subspicatus. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations (10.0, 17.8, 31.6, 56.2, and 100.0 mg test item/L (spacing factor 1.78)) of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus, over several algal generations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The study design included one control and five test material groups with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed in an open static test system. The test material concentrations were analyzed at 0 (start) and 72 hours after start of the experimental part. Defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities with an electronic particle counter. The quantification of the test item in the test samples was performed using high performance liquid chromatography (HPLC) with UV. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. The limit of quantification of the analytical method was 0.01 mg/L. The analytically determined concentrations were between 98.9 - 100.0 % at the start and 99.6 - 101.0 % at the end of exposure. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial concentrations. Therefore, the EC₅₀ values were calculated based on the nominal concentrations. Thus, the effect of the test item on freshwater green alga showed the following results: The 72-hour E_bC₅₀ was calculated to be 37.7 mg/L (95 % CI: 35.0 - 40.6 mg/L), the 72-hour E_rC₅₀ value was calculated to be 59.3 mg/L (95 % CI: 54.6 - 64.4 mg/L) and the 72-hour NOEC was determined to be 17.8 mg test item/L.

Description of key information

Under the given conditions of this study, the 72-h ErC₅₀ value was determined to be 59.3 mg/L (95 % CI: 54.6 - 64.4 mg/L) and the 72-hour NOEC was determined to be 17.8 mg test item/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

59.3 mg/L

EC10 or NOEC for freshwater algae:

17.8 mg/L

Additional information

The acute toxicity of the test item was investigated in a GLP-study according to OECD TG 201 using unicellular freshwater green alga Desmodesmus subspicatus. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations (10.0, 17.8, 31.6, 56.2, and 100.0 mg test item/L (spacing factor 1.78)) of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus, over several algal generations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The study design included one control and five test material groups with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed in an open static test system. The test material concentrations were analyzed at 0 (start) and 72 hours after start of the experimental part. Defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities with an electronic particle counter. The quantification of the test item in the test samples was performed using high performance liquid chromatography (HPLC) with UV. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. The limit of quantification of the analytical method was 0.01 mg/L. The analytically determined concentrations were between 98.9 - 100.0 % at the start and 99.6 - 101.0 % at the end of exposure. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial concentrations. Therefore, the EC₅₀ values were calculated based on the nominal concentrations. Thus, the effect of the test item on freshwater green alga showed the following results: The 72-hour E_bC₅₀ was calculated to be 37.7 mg/L (95 % CI: 35.0 - 40.6 mg/L), the 72-hour E_rC₅₀ value was calculated to be 59.3 mg/L (95 % CI: 54.6 - 64.4 mg/L) and the 72-hour NOEC was determined to be 17.8 mg test item/L.