Benzoic acid, C12-15-alkyl esters

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

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:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

July 26th, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical pre-test
- Concentrations: nominal loading rates of 10.0 mg test item/L.
- Sampling method: test solution was prepared as water-accomodated fraction, distributed to test
vessels, and incubated under test conditions. After 0h, 24h, 48h, 72h and 96h, samples were taken and measured.

Definitive test
- Concentrations: 10 mg test item/L (nominal)
- Sampling method: At test start, samples of fresh test solutions were taken from each test vessel before adding of test organisms. For aged test solutions, samples were taken also for all test vessels at the day of the first medium renewal. Thereafter, sampling was performed once per week in fresh and aged test media.
For each week, one representative vessel of control and treatment were sampled.
- Sample storage conditions before analysis: Chemical analysis of the processed samples was applied immediately after sampling. Samples which were not measured within 24 h after sampling, were immediately stored at ≤ - 18 °C until analysis.

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 10 L glass flasks with a drain port near the bottom. The flask was filled up to the volume of 10 L with dilution water to obtain the target loading of 10.0 mg test item/L. Another flask was filled with dilution water only which served as control. The test solutions was stirred slowly to avoid bubble and foam formation for about 72 h at room temperature (about 20°C). To separate insoluble parts from the aqueous phase the WAFs was left to stand to settle for at least 24 h. After settling, the first approximately 250 - 500 mL were drained from the port at the bottom and then discarded to remove any insoluble test item. The following about 8000 mL were released and used as test solution. The test solutions were freshly prepared before test start and before each renewal three times a week.
- Eluate: Purified drinking water
- Differential loading: In an analytical pre-test, which was performed in parallel for a study to assess chronic toxicity to Daphnia magna (OECD 211, EVO-073/4-21/G), it was demonstrated that the loading rate was without influence on the dissolved test item, i.e. higher loading rates did not result in higher concentrations measured. Therefore, the definitive test with D. rerio was performed as Limit test and in accordance with the OECD 210 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.):
- Other relevant information:

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain Origin: West Aquarium GmbH, 37431 Bad Lauterberg, Germany
- Source: Test facility bred

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs):
- Holding of parental fish and breeding conditions: Parental fish were held in aquaria with a total volume of 150 L. Holding water was of the same quality as used in the test (purified drinking water, see below). The maximum age for parental fish was 2 years. Stock density was approximately 1 g/L volume. Holding temperature was 26 ± 2 °C. Light/dark cycle is 12 h/12 h. Flow through rate was adjusted to reach approx. one total exchange of water per day. Fish were fed daily ad libitum with TetraMin® Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina). The brood stock was visually checked every day for mortality, illness, parasites or abnormal behavior. No prophylactic treatment of fish took place. Only healthy fish without diseases and abnormalities were used as parental fish for the production of fertilized eggs.
- Method of collection of fertilised eggs: Eggs were collected in a glass spawning-tray placed at the bottom of the holding vessels. The tray was covered with a stainless steel lattice to prevent adult fish from predating the eggs. An artificial substrate was attached to the lattice to stimulate spawning into the tray. The turn on of lighting (one neon lamp per vessel, light intensity approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel) induced mating and spawning of fish.
- Subsequent handling of eggs: Collected eggs were transferred from the spawning-tray into a sieve, rinsed with clean water in order to remove any debris and then put into glass dishes. Fertilized eggs (microscopic determination of >four cell stage, i.e. early blastula stage) were then transferred by means of a widened and de-burred pipette tip into the test chambers. Time from spawning until transferring into the test solutions was kept as short as possible.

POST-HATCH FEEDING
When hatch was finished, from day 5 dpf onwards (dpf = days post fertilization), larvae were fed at least twice daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany) and liquid rearing feed (Nobil fluid, JBL, Neuhofen, Germany). From approximately day 14 (dpf) on, brine shrimp nauplii (Artemia salina) were added twice daily. From day 14 (dpf) on, ground TetraMin flakes were added once daily to the fish feed. From approximately day 21 onwards, breeding food was exchanged by ground TetraMin flake food (Tetra Werke, Melle, Germany), ad libitum.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

35 d

Hardness:

holding and dilution water hardened with CaCl2

Test temperature:

25.8 °C to 26.6 °C

pH:

7.23 - 8.13

Dissolved oxygen:

79 % - 100 %

Salinity:

Not applicable

Conductivity:

tap water conductivity 269 - 273 µS/cm

Nominal and measured concentrations:

Nominal concentration: 10.0 mg/L
Measured concentrations: < LOQ - 60.5 µg/L in fresh samples, < LOQ - 1.44 µg/L in aged samples; geomean: 2.53 µg

Details on test conditions:

TEST SYSTEM
- Embryo cups (if used, type/material, size, fill volume): 1 fry cage with 20 eggs, per aquarium, stainless steel (ISO 3310-1) analytical sieves, diameter 10 cm, brim height 4.5 cm, bottom sieve net mesh width 355 μm, used from day 1 - 21
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass beakers with a total volume of 3 L and approx. 2 L of test medium.
- Aeration: via a needle coupled with a tube
- Renewal rate of test solution (frequency/flow rate): A complete exchange of the test media was performed three times per week.
- No. of fertilized eggs/embryos per vessel: 20 surviving fish per vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not exceeding 5 g/L fish wet weight of solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water, sourced from local district water production plants, mostly fed by small springs and percolation, purified via 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
- Hardness:
-- 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:
-- pH: before test start and before each renewal
-- Oxygen concentration: before adding eggs and afterwards at least twice per week
-- water temperature: each working day in all test vessels and in the control. Additionally, the water temperature was measured continuously in at least two control vessels.
-- Other water parameters: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours light / 12 hours dark
- Light intensity: 1000 lux, measured 5 cm above the water surface in the middle of the test vessel

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Qualitative observations on hatching and survival were made daily. Dead embryos, larvae and juvenile fish were removed as soon as observed. Observations of abnormal appearance or behavior were made daily, too. After 21 and 35 days larvae/juvenile fish were photographed and the survival rates as well as the individual total lengths (at day 35, only) of the animals were determined using digital image processing (UTHSCSA ImageTool Version 3.0; University of Texas Health Science Center at San Antonio). The total length was measured to the nearest 1 mm.
At test end, the remaining fish of each test vessel were pooled and weighted to determine the group wet weight. The fish were euthanized according to the German Animal Welfare Act. Afterwards, they were dried overnight in a cabinet dryer. The group dry weight was measured using an analytical balance. The single dry weight per fish was calculated by dividing the group dry weight by the number of surviving fish at test end.

POST-HATCH DETAILS
- Begin of post-hatch period: 6 dpf
- No. of hatched eggs (alevins)/treatment released to the test chamber: all
- Release of alevins from incubation cups to test chamber on day no.: 21

Reference substance (positive control):

no

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.35 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching success (6 dpf), Post-hatch survival, Total length, Wet weight, Dry weight, Behavioral abnormalities

Key result

Duration:

35 d

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Hatching success (6 dpf), Post-hatch survival, Total length, Wet weight, Dry weight, Behavioral abnormalities

Details on results:

- Mortality/survival at embryo and larval stages:
Mortality of larvae occurred mainly before 21 dpf, during the phase of transition from yolk sac feeding to external feeding. At 21 dpf, the post-hatch survival rate in controls reached a mean value of 93.8%. At test end, the post-hatch survival rate in controls was still at a mean value of 93.7%. The post-hatch survival in the treatment level of 2.53 µg test item/L (mean measured) was at 91.3% at 21 dpf and remained at 91.3% until 35 dpf.
- Overall mortality/survival:
- Days to hatch and numbers hatched: Hatch was completed at 6 dpf in all replicates, with no difference between the treatment and the control. The mean hatching success was 100%.
- Data for length and weight of surviving fish:
Mean total length: 15 mm in both control and test
Mean wet weight of the fish at test end: 34.9 mg in controls, 36.5 mg in test
Mean dry weight at test end: 8.75 mg in controls, 9.70 mg in test
- Type of and number with morphological abnormalities: not reported
- Type of and number with behavioural abnormalities: none
- 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
- Incidents in the course of the test which might have influenced the results: none

Reported statistics and error estimates:

For each endpoint, the NOEC (and NOEL) was determined. All statistics were calculated using ToxRat® Professional 3.3.0 [10]. For NOEC-determination, quantal data were arcsine-transformed prior to analysis. No Observed Effect Concentrations (NOEC) were calculated, using ANOVA, followed by a two-sample t-test procedure to determine if there is a significant effect between the control and the treatment (Student t-test).
Details of all statistical analyses were reported, including exact p-values for all statistical comparisons. Prior to use of parametric procedures, results of tests of normality and homogeneity of variance were considered. Failure to confirm assumptions of normality and homogeneity of variance resulted in the use of a suitable non-parametric test for the data involved. Results of all tests for normality and homogeneity of variance were reported along with the results of the parametric or non-parametric tests.
Since the test was performed as limit test, it was not possible to analyze the data by regression to determine ECx-values.

Test item concentrations

The test item showed a low solubility in the test medium with measured initial concentrations in single replicates of 2.05 to 60.5 µg/L throughout the test. During the time interval until renewal of the test solutions, test item concentrations decreased and showed measured values between <LOQ to 1.44 µg/L. 
Since measurements were not within the range of ± 20% of nominal concentrations throughout the test, the evaluation of the biological effects and the determination of the LOEC/NOEC (and LOEL/NOEL) was performed on basis of the geometric mean measured test concentration. For calculations of the geomean concentration, measured values, which were below the LOQ were set to half the LOQ (0.1 µg/L).

Geomean concentrations of fresh and aged solutions were first calculated for each sampling occasion and were <LOQ for the control and between 0.70 and 5.37 µg test item/L for the treatment level throughout the study.
Afterwards, the overall geometric mean concentration was calculated which was at 2.53 µg test item/L for the treatment level (0.0253 % of nominal).

 

 

Summary of NOEL/LOEL and NOEC/LOEC determination during the course of the study

Parameter	NOEC / LOEC Nominal loading [mg/L]	NOEC / LOEC Mean measured concentration [µg/L]
Hatching success	≥ 10 / >10	≥ 2.53 / > 2.53
Post-hatch survival at 35 dpf	≥ 10 / >10	≥ 2.53 / > 2.53
Total length at 35 dpf	≥ 10 / >10	≥ 2.53 / > 2.53
Wet weight at 35 dpf	≥ 10 / >10	≥ 2.53 / > 2.53
Dry weight at 35 dpf	≥ 10 / >10	≥ 2.53 / > 2.53

Validity of the test
The validity criteria according to the OECD Test Guideline 210 were fulfilled. These included:

• 
  

  The dissolved oxygen concentration was at least 60% of the air saturation value throughout the test.

  
  


• 
  

  The water temperature did not differ by more than ± 1.5 °C between successive days at any time during the test and was within 26 ± 1.5 °C (the test vessels were placed in one water bath).

  
  


• 
  

  The mean hatching rate in controls was greater than 70%.

  
  


• 
  

  Post-hatch success in the controls was greater than 75%.

  
  

The adherence to these criteria is listed in the table below.

Validity criteria for a Fish ELS toxicity test according to OECD TG 210

Validity criterion	Observed/measured value	Compliance
Dissolved oxygen content: >60%	Mean oxygen content between 90.1 % and 95.9 %. (Single values between 79 and 100 %)	yes
Water temperature: 26 +/- 1.5 °C	Mean water temperatures between 26.1 °C to 26.3 °C. (Single values between 25.8 and 26.6 °C)	yes
Hatching success of eggs in controls: > 70%	The mean hatching rate in controls was determined at 100%.	yes
Post-hatch survival of fish larvae in controls: ≥ 75 %	The mean post-hatch survival of fish larvae and fry in controls was determined at 93.8%.	yes
The analytical measure of the test concentrations is compulsory. A validated method for analysis of the exposure chemical with a detection limit well below the lowest nominal concentration is used to measure test concentrations.	Analytical measurements of test item concentrations were performed in fresh and aged test solutions at test start and once weekly thereafter. The recovery, efficiency and repeatability of the analytical procedure was demonstrated by procedural recoveries. Recoveries in samples of test medium fortified at LOQ level (0.20 µg/L) and at the level of concentration tested (10 000 µg/L) with a mean recovery of 0.0253%, corresponding to 2.53 µg/L.	yes     yes

Validity criteria fulfilled:

yes

Conclusions:

For all endpoints examined, no statically significant differences to the control was determined, therefore NOEC (NOEL) and LOEC (LOEL) were determined as follows:
NOEC of ≥ 2.53 µg test item/L (geometric mean measured concentration), corresponding to
NOEL of ≥ 10 mg test item/L (nominal loading).
LOEC > 2.53 µg test item/L (geometric mean measured concentration), corresponding to
LOEL > 10 mg test item/L (nominal loading).

Executive summary:

The chronic toxicity of the test item to early life stage of zebrafish (Danio rerio) was studied under static renewal conditions (according to Annex 2 of OECD guideline 210). Freshly fertilized eggs of D. rerio were exposed to the test chemical at concentrations of 10 mg/L nominal, corresponding to 2.53 µg test item/L (geometric mean measured concentration). There were no significant differences between test and control regarding hatching success (6 dpf), post-hatch survival, total length, wet weight, dry weight and behavioral abnormalities. Therefore, the 35 day NOEC value was determined to be ≥ 2.53 µg test item/L (geometric mean measured concentration), corresponding to a NOELR of ≥ 10 mg test item/L (nominal loading).
The LOEC was > 2.53 µg test item/L (geometric mean measured concentration), corresponding to a LOELR > 10 mg test item/L (nominal loading).

All validity criteria were met. This toxicity study is classified as acceptable and satisfies the guideline requirement for early life toxicity study with fish.

Description of key information

35 d NOEC ≥ 2.53 µg test item/L (geometric mean measured concentration)

35 d NOELR ≥ 10 mg test item/L(nominal)

(OECD TG 210, Danio rerio, static renewal, RL1, GLP)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 10 mg/L

Additional information

The chronic toxicity of the test item to early life stage of zebrafish (Danio rerio) was studied under static renewal conditions (according to Annex 2 of OECD guideline 210). Freshly fertilized eggs of D. rerio were exposed to the test chemical at concentrations of 10 mg/L nominal, corresponding to 2.53 µg test item/L (geometric mean measured concentration). There were no significant differences between test and control regarding hatching success (6 dpf), post-hatch survival, total length, wet weight, dry weight and behavioral abnormalities. Therefore, the 35 day NOEC value was determined to be ≥ 2.53 µg test item/L (geometric mean measured concentration), corresponding to a NOELR of ≥ 10 mg test item/L (nominal loading).
The LOEC was > 2.53 µg test item/L (geometric mean measured concentration), corresponding to a LOELR > 10 mg test item/L (nominal loading).