Benzyl benzoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/01/2013 - 11/03/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from new and old media when the media were being renewed (3 days per week).

Vehicle:

no

Details on test solutions:

A stock solution was created by adding 20mg of the test substance to 2 litres of pH adjusted dilution water and treating with an ultra-turrax for 60 seconds at 8000 rpm and stirred for 10 minutes with a magnetic stirrer. The pH was adjusted to 7.0. To create the test concentrations appropriate amounts of the stock solution were diluted with pH adjusted dilution water to a volume of 2000mL. For each replicate 100 mL of these solutions was taken. Ten replicates were prepared for each test concentration.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organisms were parthenogenetic female Daphnia magna STRAUS. They were sourced from an in-house population which is maintained at constant temperature (20±1oC) with a 16 hour light/8 hour dark photoperiod. Daphnia are fed exclusively with unicellular green algae (Desmodesmus subspicatus).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

15.8-16.8 dH

Test temperature:

19.8-20.3oC

pH:

7.0-7.7

Dissolved oxygen:

8.5-9.9

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0(control), 0.125, 0.25, 0.5, 1, 2, and 3 mg/L

Details on test conditions:

Following two range-finding test Daphnia were exposed to the test substance at nominal concentrations of 0.125, 0.25, 0.5, 1, and 3 mg/L. Single Daphnia neonates were placed in 150mL beakers with 100 mL of test solution. Ten replicates were used for each concentration. During the test the test solutions were renewed three times per week. The incubation temperature during the test was 19.8-20.3oC, with a photoperiod of 16h light: 8h dark, and a light intensity not exceeding 20 µE x m-2 x s-1.
Daphnia were fed daily with living cells of Desmodesmus subspicatus.
The criterion of adverse effects used in this study was the item-induced inhibitory effect on the parthenogenetic reproduction rate and the mobility behaviour/mortality rate of parent Daphnia, all effects being assessed at least three times a week. However, test vessels were inspected daily for the occurrence of juveniles and marked accordingly. When juveniles in these marked vessels were counted the next day (or after the weekend), the number of offspring was assigned to the previous day when juveniles were first observed.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.258 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.455 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.97 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.97 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Concentrations in the test solutions ranged from 81.0% to 92.1% of the nominal values in the freshly prepared media and from 1.0% to 46..8% in the media after 48 or 72 hours exposure. As such, endpoints were calculated based on time weighted average values.
The 21-day NOEC for reproduction was calculated to be 0.258 mg/L , and the LOEC was calculated to be 0.455 mg/L.
For mortality the 21-day NOEC was calculated to be ≥ 0.97 mg/L, and the LOEC > 0.97 mg/L.

Reported statistics and error estimates:

The reproductive output of the Daphnia exposed to the test item was compared to that of the control in order to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) according to Williams Multiple Sequential t-Test Procedure and to Fisher`s Test with Bonferroni Correction.

Table 1: Mean reproduction rate

Test item concentration nominal [mg/L]	Time-weighted mean measured concentration [mg/L]	Mean reproduction rate	SD	CV [%]
Control	----	95.3 [10]	9.7	10.2
0.125	-----	97.1 [10]	9.5	9.8
0.25	-----	102.3 [9]	9.8	9.5
0.5	0.123	98.7 [9]	21.8	22.1
1	0.258	91.5 [10]	20.3	22.1
2	0.455	69.6 [9]	22.9	32.9
3	0.970	29.1 [8]	17.1	58.5

Table 18- Immobilisation/mortality rate and mean reproduction rate of parent Daphnia

Test item concen-tration nominal	Time-weighted mean measured concentration [mg/L]	Immobilisation/ mortality rate of parentDaphnia		Mean reproduction rate of parentDaphnia
[mg/L]		absolute	%	absolute	%
Control	----	0	0	95.3 [10]	100
0.125	-----	0	0	97.1 [10]	101.9
0.25	-----	1	10	102.3 [9]	107.3
0.5	0.123	1	10	98.7 [9]	103.6
1	0.258	0	0	91.5 [10]	96.0
2	0.455	1	10	69.6 [9]	73.0
3	0.970	2	20	29.1 [8]	30.5

Table 8- Abiotic parameters of control

Day	Date 2013	Parameter	Replicate 1 (new medium)	Day	Date 2013	Parameter	Replicate 1 (old medium)
0	01-16	pH value	7.2	2	01-18	pH value	7.7
0	01-16	O2[mg/L]	8.8	2	01-18	O2[mg/L]	8.9
0	01-16	O2[%]	98	2	01-18	O2[%]	100
0	01-16	t [°C]	20.9	2	01-18	t [°C]	20.1
0	01-16	[°dH]	16.2	2	01-18	[°dH]	16.4
7	01-23	pH value	7.1	9	01-25	pH value	7.3
7	01-23	O2[mg/L]	8.7	9	01-25	O2[mg/L]	9.4
7	01-23	O2[%]	97	9	01-25	O2[%]	101
7	01-23	t [°C]	20.8	9	01-25	t [°C]	19.8
7	01-23	[°dH]	16.1	9	01-25	[°dH]	16.5
16	02-01	pH value	7.0	19	02-04	pH value	7.3
16	02-01	O2[mg/L]	8.6	19	02-04	O2[mg/L]	8.8
16	02-01	O2[%]	96	19	02-04	O2[%]	95
16	02-01	t [°C]	20.8	19	02-04	t [°C]	19.8
16	02-01	[°dH]	15.8	19	02-04	[°dH]	16.1

Table 9- Abiotic parameters 3mg/L (nominal)

Day	Date 2013	Parameter	Replicate 1 (new medium)	Day	Date 2013	Parameter	Replicate 1 (old medium)
0	01-16	pH value	7.2	2	01-18	pH value	7.6
0	01-16	O2[mg/L]	8.6	2	01-18	O2[mg/L]	8.7
0	01-16	O2[%]	99	2	01-18	O2[%]	96
0	01-16	t [°C]	21.1	2	01-18	t [°C]	20.3
0	01-16	[°dH]	16.2	2	01-18	[°dH]	16.3
7	01-23	pH value	7.1	9	01-25	pH value	7.6
7	01-23	O2[mg/L]	8.7	9	01-25	O2[mg/L]	9.9
7	01-23	O2[%]	98	9	01-25	O2[%]	105
7	01-23	t [°C]	21.0	9	01-25	t [°C]	19.4
7	01-23	[°dH]	16.8	9	01-25	[°dH]	16.4
16	02-01	pH value	7.0	19	02-04	pH value	7.5
16	02-01	O2[mg/L]	8.5	19	02-04	O2[mg/L]	8.9
16	02-01	O2[%]	97	19	02-04	O2[%]	97
16	02-01	t [°C]	21.0	19	02-04	t [°C]	19.7
16	02-01	[°dH]	15.8	19	02-04	[°dH]	16.1

Validity criteria fulfilled:

yes

Conclusions:

The 21-day NOEC for reproduction was determined to be 0.258 and the 21-day LOEC for reproduction was determined to be 0.455 mg/L.

Executive summary:

A 21 -day test was conducted to assess the effects of benzyl benzoate on the reproductive output of parthenogenetic Daphnia magna under semi static conditions. Female Daphnia magna neonates were exposed to the test substance at concentrations of 0.125, 0.25, 0.5, 2, and 3 mg/L. Test organisms were kept under constant light and temperature, and test media was renewed 3 times per week. The number of offspring produced was recorded throughout the test. The number of mortalities was also recorded.

After 21 -days exposure, the total number of living offspring produced per parent animal alive at the end of the test was assessed.

The 21 -day NOEC for reproduction was determined to be 0.258 mg/L based on time-weighted average concentrations. The 21 -day LOEC for reproduction was determined to be 0.455 mg/L based on time-weighted average concentrations.

Description of key information

The 21-day NOEC for reproduction in Daphnia magna was determined to be 0.258 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.258 mg/L

Additional information

A 21 -day test was conducted to assess the effects of benzyl benzoate on the reproductive output of parthenogenetic Daphnia magna under semi static conditions. Female Daphnia magna neonates were exposed to the test substance at concentrations of 0.125, 0.25, 0.5, 2, and 3 mg/L. Test organisms were kept under constant light and temperature, and test media was renewed 3 times per week. The number of offspring produced was recorded throughout the test. The number of mortalities was also recorded.

After 21 -days exposure, the total number of living offspring produced per parent animal alive at the end of the test was assessed.

The 21 -day NOEC for reproduction was determined to be 0.258 mg/L based on time-weighted average concentrations. The 21 -day LOEC for reproduction was determined to be 0.455 mg/L based on time-weighted average concentrations.