Benzoic acid, C12-15-alkyl esters

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:

2020 - 2021

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)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

02.10.2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical pre-test
- Concentrations: nominal loading rates of 1.0, 10.0 and 100 mg test item/L. The nominal concentration of the main test was 10 mg test item/L.
- Sampling method: test solutions were prepared as water-accomodated fractions, distributed to test vessels, and incubated under test conditions. After 0h, 24h, 48h and 72h, samples were taken and measured.

Definitive test with D. magna
- Concentrations: 10 mg test item/L (nominal)
- Sampling method: Sampling of test solutions was performed at each media renewal (three times per week). Samples of fresh test solutions were taken from the test solution preparations before adding daphnids. For aged test solutions, samples were taken from a representative test vessel per treatment.
- Sample storage conditions before analysis: Chemical analysis of the processed samples was applied immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test solution was prepared as individual Water Accommodated Fraction (WAF) according to the OECD guidance No. 23. An appropriate amount of the test item was weighed and transferred into a 2 L brown glass flasks with a drain port near the bottom. The flask was filled up to the volume of 2.0 L with test medium to obtain the target loading of 10.0 mg test item/L. Another flask was filled with test medium only which served for the control. The test media was stirred slowly to avoid bubble and foam formation for about 72 h at room temperature (about 20°C). After 24h settling time, oily droplets were visible at the water surface. The first approximately 150 mL were drained from the port at the bottom and discarded. The following about 1000 mL were released and used as test solution. The remaining part of the WAF solution were not used.
The test solutions were freshly prepared before test start and before each renewal three times a week
- Eluate: Purified, Cu-reduced tap water was used as holding- and dilution water. Purification includes filtration with activated charcoal and aeration.
- Differential loading: An analytical pre-test was performed with three nominal loading rates of 1.0, 10.0 and 100 mg test item/L, prepared as WAF. Test solutions were incubated in 50 ml test vessels under test conditions. After 0h, 24h, 48h and 72h, samples were taken and measured. No grading was observed between the loading rates. The measured test concentrations were 2.68 – 4.30 at 0 h and decreased during the 72 h time interval but were mostly above the target LOQ of 0.2 µg/L. As the dissolved test item concentration was not influenced by the loading rate, the definitive test with Daphnia magna was performed as Limit test with one nominal loading rate (10.0 mg/L).
- Controls: The control consisted of dilution water only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): oily droplets were visible at the water surface of the 2 L WAF. The presence of such droplets in the test vessels was not reported.
- Other relevant information:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: freshwater flea
- Age at study initiation (mean and range, SD): 4 – 24 hours old
- Weight at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Stage and instar at study initiation:
- Method of breeding:
- Source: Origin of the cladocerans is the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test are bred in the laboratory of the Fraunhofer IME for over 15 years.
- Age of parental stock (mean and range, SD): at least 3 weeks old
- Feeding during test
-- Food type: suspensions of unicellular alga Desmodesmus subspicatus.
-- Amount and Frequency: The content of food in the test suspensions, measured at 585 nm, was kept constant at 0.2 mg C/Daphnia * Day.

ACCLIMATION
- Acclimation period: Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. Newborn D. magna were separated by sieving, the first generation was discarded.
- Acclimation conditions (same as test or not): same though kept at room temperature (temperature not recorded).
- Type and amount of food: algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL GmbH & Co. KG). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 5 mL of this suspension was given to about 1.8 L Daphnia medium.
- Feeding frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Newborn daphnia were separated by sieving. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Hardness of the Cu-reduced water was below the recommended range (>140 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Therefore, the dilution water was enriched with an appropriate amount of CaCl¬2 to increase the hardness, resulting in values of about 250 – 350 mg CaCO3/L.

Test temperature:

19.5 – 20.0 °C [permitted range: 18 – 22 °C]

pH:

The pH was within a range of 6 – 9 in fresh test solutions (7.50 – 8.12). In aged test solutions, the pH was between 8.16 and 9.37.

Dissolved oxygen:

7.23 mg/L and 11.6 mg/L

Salinity:

Not applicable

Conductivity:

tap water: 269 - 273 µS/cm

Nominal and measured concentrations:

Nominal concentration: 10.0 mg test item/L.
The test item showed a low solubility in the test medium, with measured initial concentrations of 0.1 to 13.2 µg/L. At three sampling points (day 9, 12 and 16) initial concentrations were below the LOQ. Accordingly the recovery rates of the test item in the fresh test solutions were very low with 0.001 – 0.132 % of nominal concentrations. During the time interval until renewal of the test solutions, test item concentrations decreased and showed measured values between

Details on test conditions:

TEST SYSTEM
- Test vessel
-- Type (delete if not applicable): Beakers covered with glass panes to prevent evaporation as much as possible, but also permit gaseous exchange between medium and atmosphere and access of light.
-- Material, size, headspace, fill volume: 50 – 100 mL of test solution in numbered glass beakers
-- Aeration: no active aeration
- Renewal rate of test solution (frequency/flow rate): Test solutions were exchanged three times per week. After each renewal of the test solution, fresh test solutions were distributed to the replicate beakers and the test specimens were added with a bore Pasteur pipette.
- No. of organisms per vessel: 1 young female Daphnia per vial
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.2 mg C/Daphnia * Day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified, Cu-reduced tap water was used as holding- and dilution water. The purification includes filtration with activated charcoal and aeration.
- Total organic carbon: NPOC (non-purgeable organic carbon)[mg/L] Nov 2020: 0.753, Dec 2020:0.909 mg/L
- Particulate matter:
- Metals:
-- Cd [µg/L] Nov 2020: -, Dec 2020: < 0.003 (LOD) - < 0.009 (LOQ)
-- Cr [µg/L] Nov 2020: < 0.053 (LOQ) , Dec 2020: 0.070
-- Cu [µg/L] Nov 2020: 0.871 – 0.935 , Dec 2020: 0.537 – 0.642
-- Fe [µg/L] Nov 2020: < 1.49 (LOQ), Dec 2020: 1.48 – 2.01
-- Mn [µg/L] Nov 2020: 0.106 – 0.111, Dec 2020: 0.283 – 0.307
-- Ni [µg/L] Nov 2020: 0.200 – 0.204, Dec 2020: 0.187 – 0.196
-- Pb [µg/L] Nov 2020: 0.056 – 0.061, Dec 2020: 0.015 – 0.016
-- Zn [µg/L] Nov 2020: < 2.06 (LOQ) - 6.19, Dec 2020: 1.254 – 1.267
- Pesticides:
- Chlorine: Cl [mg/L] Nov 2020:0.03, Dec 2020: 0.04
- Alkalinity: Alkalinity [mmol/L] Nov 2020: 1.8, Dec 2020: 1.6
- Ca/mg ratio: Hardness of the Cu-reduced water was below the recommended range (>140 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Therefore, the dilution water was enriched with an appropriate amount of CaCl2 to increase the hardness, resulting in values of about 250 – 350 mg CaCO3/L.
-- Tot. Hardness [mmol/L] Nov 2020: 1.0, Dec 2020: 1.2
-- Ca-hardness [mmol/L] Nov 2020: 0.9, Dec 2020: 0.9
-- Mg-hardness [mmol/L] Nov 2020: 0.1, Dec 2020: 0.3
- Conductivity [µS/cm] Nov 2020: 273, Dec 2020: 269
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen concentration, pH value, and temperature of the test solutions was checked directly before adding the animals and at each water renewal in new and aged test solutions. Hardness was checked in the control and the loading rate once per week. Other tap water parameters were checked monthly

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: 755 – 903 lx (corresponding to 10.1 – 12.0 µE/(m² *s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The numbers of immobile daphnids were visually determined daily and the immobile daphnids were removed. Any abnormalities in appearance and behavior were recorded.
The newborn daphnids per beaker were counted and removed daily until all daphnids had started to reproduce. After that, newborn daphnids were counted and removed three times per week at each water renewal. Abnormalities in condition (including male sex of adults) or presence of winter eggs were checked and recorded, if occurred. At study termination, length of the adults was measured by digital photography and image analysis and compared with the equally measured control animals.

TEST CONCENTRATIONS
An analytical pre-test was performed with three nominal loading rates of 1.0, 10.0 and 100 mg test item/L, prepared as WAF. Test solutions were incubated in 50 ml test vessels under test conditions. After 0h, 24h, 48h and 72h, samples were taken and measured. No grading was observed between the loading rates. The measured test concentrations were 2.68 – 4.30 at 0 h and decreased during the 72 h time interval but were mostly above the target LOQ of 0.2 µg/L. As the dissolved test item concentration was not influenced by the loading rate, the definitive test with Daphnia magna was performed as Limit test with one nominal loading rate (10.0 mg/L).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 1.12 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

other: reproduction of survived and introduced parent daphnids, immobilisation of parent daphnids, length at test end, age of first reproduction, development rate and intrinsic rate r

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction of survived and introduced parent daphnids, immobilisation of parent daphnids, length at test end, age of first reproduction, development rate and intrinsic rate r

Remarks on result:

other: test concentration above the water solubility

Details on results:

- Behavioural abnormalities: none (test group and control group)
- Observations on body length and weight: Daphnids showed a mean size of 4.71 mm in the control and 4.64 mm in the treatment level.
- Other biological observations: The cumulative offspring per survived and introduced parent was at 109.5 in the control and at 132.3 in the treatment level. Age at the first brood was at 10.6 days for the control and at 9.8 days for the treatment level. The development rate was at 0.095 in the control and at 0.102 in the tested treatment level. The intrinsic rate was at 0.331 in the control and at 0.356 in the tested treatment level.
No significant effects were determined.
- Mortality of control: No mortality of parental and control animals during the test period
- Other adverse effects control: none
- Immobilisation of control: none
- Abnormal responses: none (test group and control group)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: After 24h settling of the WAF, oily droplets were visible at the water surface
- Effect concentrations exceeding solubility of substance in test medium: yes, based on the results of the preliminary test the definitive study was performed as limit test with a limit concentration of 10.0 mg/L above the water solubility level (2.47 µg/L) and > LOQ

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: In order to prove the validity of the test system and test conditions at the testing facility, acute immobilization tests with K2Cr2O7 are performed regularly, as proposed by OECD 202. The latest reference study (Nov 2020) indicated a 24 h Immobilization EC50 value of 0.753 mg/L (95% CL: 0.660 – 0.856 mg/L), in agreement with the range given in OECD 211 (0.6 - 2.1 mg/L).

Reported statistics and error estimates:

For each endpoint, the NOEC and LOEC was determined.
The computer software ToxRat® was used for statistical evaluations.
The evaluation was based on the mean measured concentration (time weighted mean), since the measured concentration varied by more than 20% from the nominal loading rate throughout the test.

Reproduction data during the 21-day exposure to the test item based on the TWM concentration of the test item

TWM Conc.	Nominal Loading	Cumulative offspring per survived parent	Cumulative offspring per introduced parent	Age of first brood	Development rate	Intrinsic rate population increase
[µg/L]	[µg/L]	Mean ± SD [Ind.]	Mean ± SD [Ind.]	Mean ± SD [days]	Mean ± SD [Ind.]	Mean ± SD [Ind.]
Control	Control	109.5 ± 8.42	109.5 ± 8.42	10.6 ± 0.57	0.095 ± 0.0058	0.331 ± 0.0177
1.12	10 000	132.3 ± 16.5 (-)	132.3 ± 16.5 (-)	9.80 ± 0.48 (-)	0.102 ± 0.0048 (-)	0.356 ± 0.0072 (-)

(-) no statistically significant difference between control and treatments; SD: standard deviation

 

NOEL and LOEL values based on the TWM concentration of the test item and the nominal loading rate of the test item for the exposure of Daphnia magna for 21 days

Parameter	LOEC	NOEC	LOEL	NOEL
	TWM [µg/L]		Nominal Loading rate [mg/L]
Offspring per survived parent ^1)	> 1.12	≥ 1.12	> 10.0	≥ 10.0
Offspring per introduced parent	> 1.12	≥ 1.12	> 10.0	≥ 10.0
Immobility	> 1.12	≥ 1.12	> 10.0	≥ 10.0
Length	> 1.12	≥ 1.12	> 10.0	≥ 10.0
Age of first reproduction	> 1.12	≥ 1.12	> 10.0	≥ 10.0
Development rate	> 1.12	≥ 1.12	> 10.0	≥ 10.0
Intrinsic rate	> 1.12	≥ 1.12	> 10.0	≥ 10.0

1) No trend on immobility was observed. Therefore, according to OECD 211, it is required to report the NOEC for the cumulative offspring per survived parent. Nevertheless, results for reproduction based on the cumulative offspring per introduced parent daphnid are also presented.

 

Validity of the test
The test fulfils all validity criteria of the OECD guideline 211 as:

• Mortality in controls (0%) did not exceed 20%.
• The mean number of offspring in the control within the 21 days (109.5) was above the criterion of 60 / female.

In addition, the following quality criteria indicated in the OECD guideline were fulfilled:
• The dissolved oxygen concentration was above 3 mg/l at the beginning and during the test.
• Hardness in terms of CaCO3 was above 140 mg/L
• The pH was within a range of 6 – 9 in fresh test solutions (7.50 – 8.12). In aged test solutions, the pH was between 8.16 and 9.37.

OECD 211 test guideline states as an additional recommendation that the coefficient of variation around the mean number of living offspring produced per parent animal in the control(s) should be ≤ 25%. In the present test, the coefficient was at 7.7 %.

Validity criteria fulfilled:

yes

Conclusions:

The test organisms Daphnia magna showed no significant effects after the 21-day exposure period to the test item in terms of reproduction, immobilisation, length, age of first reproduction, development rate and intrinsic rate r of population increase.

For each endpoint considered, the NOEC was set to be higher or equal than the tested concentration and is therefore determined to be ≥ 1.12 µg/L (measured), corresponding to ≥ 10 mg/L nominal loading. The LOEC was determined at > 1.12 µg/L (measured), corresponding to > 10 mg/L nominal loading.

Executive summary:

The influence of the test item on the reproduction of Daphnia magna was investigated in a limit test with one loading rate during a 21-day semi-static exposure according to the OECD guideline 211.
Effects on the reproductive performance, survival, growth (adult length at test termination), development rate and intrinsic rate r of population increase were investigated. The test item concentrations in the treatment level were measured in aqueous samples at fresh and representative aged test solutions three times per week at media renewal.
Due to the low water solubility of the test item, the test solution, with a nominal loading rate of 10 mg test item/L, was prepared as Water Accommodated Fraction (WAF) according to the OECD guidance No. 23 in dilution water (tap water).
The evaluation of the results was based on the time weighted mean (TWM) concentration of the test item in the treatment level, calculated to be 1.12 µg/L (0.011 % of nominal).
After 21 days, mortality was 0% in both control and treatment level. The cumulative offspring per survived and introduced parent was at 109.5 in the control and at 132.3 in the treatment level.
No statistical significant effect was observed for all investigated endpoints.
The NOEC was determined to be ≥ 1.12 µg/L (measured), corresponding to ≥ 10 mg/L nominal loading. The LOEC was determined at > 1.12 µg/L (measured), corresponding to > 10 mg/L nominal loading.

Description of key information

21 d NOEC ≥ 1.12 µg/L (TWA)

21 d NOELR ≥ 10 mg/L (nominal)

(OECDE TG 211, Daphnia magna, semi-static, RL1, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 10 mg/L

Additional information

The influence of the test item on the reproduction of Daphnia magna was investigated in a limit test with one loading rate during a 21-day semi-static exposure according to the OECD guideline 211.
Effects on the reproductive performance, survival, growth (adult length at test termination), development rate and intrinsic rate r of population increase were investigated. The test item concentrations in the treatment level were measured in aqueous samples at fresh and representative aged test solutions three times per week at media renewal.
Due to the low water solubility of the test item, the test solution, with a nominal loading rate of 10 mg test item/L, was prepared as Water Accommodated Fraction (WAF) according to the OECD guidance No. 23 in dilution water (tap water).
The evaluation of the results was based on the time weighted mean (TWM) concentration of the test item in the treatment level, calculated to be 1.12 µg/L (0.011 % of nominal).
After 21 days, mortality was 0% in both control and treatment level. The cumulative offspring per survived and introduced parent was at 109.5 in the control and at 132.3 in the treatment level.
No statistical significant effect was observed for all investigated endpoints.
The NOEC was determined to be ≥ 1.12 µg/L (measured), corresponding to ≥ 10 mg/L nominal loading. The LOEC was determined at > 1.12 µg/L (measured), corresponding to > 10 mg/L nominal loading.