Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

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:

2019-05-02 to 2019-08-12

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:

2013

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 1802000003
CAS No.: 186817-80-1
Purity (certified): 98 %

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples of test media including solvent control group and control group were taken from alternating test replicates on days -2, -1 and during the exposure on study days 0, 6, 13, 20, 27 and 33. Additional analyses of the nominal concentration of 5.00 mg/L were carried out from several replicates on study days 21, 22, 23 and 26. Since an application malfunction in replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate will not be included in any evaluation and statistical analysis. The stock solution was sampled and analysed from freshly prepared and corresponding 4 days aged stock solution of one application interval on study days 9 and 13.
- Sampling method: At each sampling day 2 samples were taken per (alternating) test replicate. For each sample 10 mL of each test item concentration, the solvent control and the control were filled into 20 mL vials.
- Sample storage conditions before analysis: All samples were immediately extracted, and the extracts were stored at room temperature if necessary. Until the start of the analysis the prepared samples were stored on an autosampler.

Vehicle:

yes

Remarks:

methanol (MeOH)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
- Method: A stock solution of 200 g/L was prepared in MeOH. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the solvent into a glass flask. The solution was agitated until it was visually clear. Further stock solutions were prepared by dilution with MeOH. The stock solutions were prepared in appropriate intervals of 3 to 4 days. Syringes were filled with the freshly prepared stock solutions or pure MeOH for the solvent control every 3 to 4 days.
A flow-through exposure design was carried out. Membrane piston pumps provided the water flow. Precision syringe pumps were used for the introduction of stock solutions of the test item dissolved in methanol into the mixing chamber. The stock solutions and the dilution water were mixed in a mixing chamber (approx. volume 0.7 L) by magnetic stirring before passing the test aquaria (approx. volume 7.5 L) where the eggs/fish will be exposed. One mixing chamber was used for one test aquaria (replicate). Four replicates were set up for each test concentration, control and solvent control, respectively.
The accuracy of the water flow was checked prior to start of the exposure and three times per week thereafter.
Water exchange in the test aquaria was about 10 test vessel volumes per day (3.125 L/h).
An equilibration period of 12 days was carried out prior to start of the exposure until measured concentrations were within the range of ± 20 % of the nominal concentration.
- Control: Dilution water (without test item)
- Solvent control: Additionally, a solvent control with 0.05 mL methanol/L dilution water was prepared and tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): With regard to solubility of the test item in water methanol (MeOH) was used as a solvent. The solvent concentration was the same in each test concentration and the solvent control (0.05 mL/L). Methanol (Merck, hypergrade, batch 10992535907, I1000435910 and I1009735916).
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.05 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Not reported
- Source: All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): 15–35 adult zebrafish were kept in at least 3 separate aquaria. The fish were healthy with a mortality rate < 5 % during the last 7 days and not medically treated for at least 7 days.
- Method of collection of fertilised eggs: About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless- steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After approximately 1 hour the glass dishes were gently removed.
- Subsequent handling of eggs: Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded. About 800 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings. At least 70 % of the eggs appeared healthy. The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs). Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers can be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2-cell stage were regarded as not fertilized and will be discarded. The fertilization rate was 90 %.
Only fertilized eggs with more than 2 cells were placed directly in the test vessels. 20 eggs were introduced by random per replicate (corresponding to 80 eggs per treatment group). Eggs were less than 24 h old at experimental starting. The distribution of eggs to the concentration groups was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contain the necessary number of eggs.

POST-HATCH FEEDING
- Start date: study day 5 to 35; feeding started 3 days after the beginning of hatch (on study day 5 (post-hatch day 0)).
- Type/source of feed: Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2–5 times daily). About 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (2–6 times daily). Brine shrimp nauplii origin, breeding conditions:
Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D 67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/L, ca. 2 g eggs to 1 L salt water, gentle aeration for 24–48 hours at approx. 22 °C). 24–48 h old brine shrimp nauplii were harvested, washed in a stainless- steel mesh and resuspended in tap water.
- Amount given: Ad libitum
- Frequency of feeding: Food ST-1 and fine milled brine shrimp nauplii: 2–5 times daily. About 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (2–6 times daily).

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Remarks on exposure duration:

35 days (30 days post hatch), depending on post-hatch day 0 (study day 5).

Hardness:

53 to 75 mg CaCO3/L

Test temperature:

26 ± 1.5 °C

pH:

7.60 to 8.28

Dissolved oxygen:

76 to 100 % ASV

Nominal and measured concentrations:

nominal: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
time-weighted arithmetic mean measured concentrations: 0.640 – 1.29 – 2.33 – 4.60 – 9.61 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used. Test vessels were covered by glass lids. The volume of the test media is approximately 7.5 L.
- Cleaning: The test vessels were siphoned as needed to remove excess fecal material and uneaten food, also to minimize microbial growth and biodegradation of the test compound. Cleaning started on study day 4. Furthermore, the mesh coated fittings were cleaned once per day from study day 4 until the end on the test.
- Aeration: The dilution water supply tank was aerated. No additional aeration of the test vessels was provided.

- Type of flow-through (e.g. peristaltic or proportional diluter): A flow-through exposure design was carried out. Membrane piston pumps provided the water flow.
- Renewal rate of test solution (frequency/flow rate): Water exchange in the test aquaria was about 10 test vessel volumes per day (3.125 L/h).
- No. of fertilized eggs/embryos per vessel: Four replicates per test concentration and control, with 20 eggs each (80 eggs per test concentration, solvent control and control)
For the whole study (including the range finding test) 680 eggs/fish were used.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was maintained.

TEST MEDIUM/WATER PARAMETERS
- Source/preparation of dilution water: Tap water was used for testing. The water was filtered on activated charcoal to remove residual chlorine.
- Total organic carbon: The total organic carbon (TOC) sampled from the dilution water supply tank was determined on study days 2, 7, 14, 21, 29 and 33. Mean TOC was 1.57 mg/L throughout exposure.
- Chlorine: Residual chlorine, measured from the dilution water supply tank on study days 1, 7, 14, 21, 28 and 33 was < 0.01 mg/L.
- Alkalinity: 0.5 mmol/L (recent measurement: 2019-04-04)
- Salinity: freshwater
- Culture medium different from test medium: same
- Intervals of water quality measurement:
Once per hour: Temperature in the dilution water, measured in one control vessel
At least 3 times per week: Determination of…
a) Dissolved oxygen in all replicates of each test group
b) Check of flow rates of the test media (variation < 10% throughout exposure)
Weekly:
a) pH-value and temperature in all replicates of each test group
b) TOC and Chlorine from dilution water
c) Total hardness in one replicate of control, solvent control and
d) the limit concentration
The light intensity on the surface of the test aquaria was measured at the start of the exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark cycle (2 transition periods, 30 minutes each). Diffuse light (7–750 lux on water surface).
- Light intensity: Light intensity was measured at the start of exposure on the surface of all test vessels and ranged from 182 to 386 lux (mean: 254 lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Hatching, mortality, abnormal appearance and behaviour, fish size, fish weight
All biological parameters were observed daily.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: A solvent control and two test concentrations of the test item of 1 and 10 mg/L were tested. Two replicates per solvent control and test concentration were tested. 20 eggs per replicate were exposed to each concentration level and the solvent control.
- Results used to determine the conditions for the definitive study: Yes

POST-HATCH DETAILS
- Begin of post-hatch period: study day 5 = post-hatch day 0. Hatch began on study day 2 in the control, solvent control and the lowest test item concentration and continued until study day 10. The hatch of the control, solvent control and the nominal test item concentrations 0.625 to 2.50 mg/L was completed until study day 7. The hatch completion in the test concentrations of 5.00 and 10.0 mg/L was delayed and not finished before study day 10. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of both 95% in the control and solvent control
- Fry survival (post-hatch survival): The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75 %). The fry survival (post-hatch survival) at the end of the study was 76 % in the control and 85 % in the solvent control. A concentration related decrease of the post hatch survival was detected with increasing test concentrations.

Reference substance (positive control):

no

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.61 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 9.61 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

35 d

Dose descriptor:

EC10

Basis for effect:

number hatched

Remarks on result:

not determinable

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

1.29 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

2.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

2.57 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

8.04 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

1.29 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

2.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

1.86 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

4.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

2.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

35 d

Dose descriptor:

EC10

Basis for effect:

mortality

Remarks on result:

not determinable

Duration:

35 d

Dose descriptor:

EC50

Effect conc.:

4.47 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages:
Fry Survival
The post-hatch survival in the control replicates met the validity criteria of the guideline (required: ≥ 75 %). The fry survival (post-hatch survival) at the end of the study was 76 % in the control and 85 % in the solvent control. A concentration related decrease of the post hatch survival was detected with increasing test concentrations. The step-down Cochran-Armitage Test (alpha = 0.05) was performed for statistical analysis of post hatch survival data on study day 35 (PHD 30). Statistically significant differences were found between the control groups and the time-weighted arithmetic mean measured concentrations of 4.60 and 9.61 mg/L.
The NOEC and the LOEC for this endpoint were 2.33 mg/L and 4.60 mg/L, respectively.
The LC50 value for post hatch survival on study day 35 (PHD30) was 4.34 mg/L.

Overall Survival (Overall Mortality)
The cumulative survival at the end of the exposure, related to the number of eggs introduced on day 0 was 74 % in the control group and 81 % in the solvent control group. A concentration related decrease of the overall survival was detected with increasing test concentrations.
The step-down Cochran-Armitage test (alpha = 0.05) was performed for statistical analysis of overall survival data on study day 35 (PHD 30). Statistically significant differences were found between the control groups and the time-weighted arithmetic mean measured concentrations of 4.60 and 9.61 mg/L.
The NOEC and the LOEC for this endpoint were 2.33 mg/L and 4.60 mg/L, respectively.
The LC50 value for overall survival on study day 35 (PHD30) was 4.47 mg/L.

- Days to hatch: Hatch began on study day 2 in the control, solvent control and the lowest test item concentration and continued until study day 10. The hatch of the control, solvent control and the nominal test item concentrations 0.625 to 2.50 mg/L was completed until study day 7. The hatch completion in the test concentrations of 5.00 and 10.0 mg/L was delayed and not finished before study day 10. Study day 5 was determined to be post hatch day 0 (PHD 0) with a hatching rate of both 95 % in the control and solvent control.

Statistical procedures were applied for the total number of test organisms that have hatched on study days 4, 5, 6 and 10. The Chi² 2 × 2 test (alpha = 0.05) was performed for statistical analysis of hatch. Statistically significant differences were found between the controls and the test concentrations of 4.60 and 9.61 mg/L on study days 4, 5 and 6. On study day 10 no statistically significant differences were detected.
The NOEC and the LOEC (based on time-weighted arithmetic mean measured test item concentration) for this endpoint were determined to be ≥ 9.61 mg/L and > 9.61 mg/L, respectively.

- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: see “Any other information on results incl. tables”.
- Number of fish in swim-up stage at one or more time periods (e.g., day ×1, ×2): Swim-up was observed for a 3-day period from study days 4 to 6 (see Table 5). Newly hatched fry began to swim up on study day 4 (PHD -1). On study day 6 (PHD 1), all surviving larvae had swum up. The swim-up behaviour in the highest test concentration was not completely evaluable, since behavioural effects caused by the presence of the test item interfered with the swim-up. No statistical analysis of swim-up data was carried out.

- Observations on body length and weight of young and/or exposed parents at one or more time periods: Fry growth, expressed as length and wet weight, was measured on study day 35 (PHD 30) from all survivors. The coefficients of variations (length: between and within replicates, weight: between replicates) well met the 90th percentiles coefficients of variations as stated in the guideline.
With the statistical procedures applied for fresh weight data (Williams multiple sequential t-test procedure, alpha = 0.05) the time-weighted arithmetic mean measured concentrations of 2.33 to 9.61 mg/L showed significant differences for this parameter.
For the statistical analysis of the mean total length of all survivors a multiple sequentially rejective Welsh-t-test after Bonferroni Holm was performed (alpha = 0.05). A statistically significant difference was found for the time-weighted arithmetic mean measured concentration of 2.33 mg/L.
Therefore, the NOEC and the LOEC (time-weighted arithmetic mean measured test item concentrations) for length and wet weight were laid down as 1.29 mg/L and 2.33 mg/L, respectively.
- Morphological and Behavioral Effects: No biologically significant morphological and behavioral effects were observed in the control, the solvent control and the test item concentrations of 0.640 to 2.33 mg/L. In the two highest test concentrations biologically significant behavioral effects as quiescence (Q), lethargy (L), unusually long arresting on the ground (G), side position (S) and tumbling (T) were observed from study day 6 up to study day 22.
- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: not reported

For further details refer to “any other information on results incl. tables”.

Hatch/Hatching time

Table 1: Hatch/hatching time (control, solvent control and test item concentrations)

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	PHD -2	PHD -1	PHD 0	PHD 1	PHD 2	PHD 3	PHD 4	PHD 5
		Study day 3	Study day 4	Study day 5	Study day 6	Study day 7	Study day 8	Study day 9	Study day 10
		Hatching rate [%] per study day
Control	1	20	90	95	100	100	100	100	100
	2	35	90	90	90	90	90	90	90
	3	10	75	95	100	100	100	100	100
	4	35	85	100	100	100	100	100	100
	Mean	25	85	95	98	98	98	98	98
Solvent control	1	55	100	100	100	100	100	100	100
	2	45	95	95	100	100	100	100	100
	3	25	90	95	95	95	95	95	95
	4	50	90	90	90	90	90	90	90
	Mean	44	94	95	96	96	96	96	96
0.640	1	90	95	95	95	95	95	95	95
	2	80	90	100	100	100	100	100	100
	3	40	95	95	95	95	95	95	95
	4	40	100	100	100	100	100	100	100
	Mean	63	95 (-)	98 (-)	98 (-)	98	98	98	98 (-)
1.29	1	60	95	95	95	95	95	95	95
	2	70	95	95	95	95	95	95	95
	3	70	85	85	85	85	85	85	85
	4	45	90	95	95	95	95	95	95
	Mean	61	91 (-)	92 (-)	93 (-)	93	93	93	93 (-)
2.33	1	65	90	95	95	95	95	95	95
	2	25	90	90	90	100	100	100	100
	3	20	80	100	100	100	100	100	100
	4	15	85	90	95	95	95	95	95
	Mean	31	86 (-)	94 (-)	95 (-)	98	98	98	98 (-)
4.60	1	40	95	95	100	100	100	100	100
	2	0	55	85	95	100	100	100	100
	3	20	65	80	80	80	85	85	85
	4	15	60	90	90	90	90	95	100
	Mean	12	60 (+)	85 (+)	88 (+)	90	92	93	95 (-)
9.61	1	35	80	85	85	85	85	90	90
	2	15	65	80	80	85	90	95	95
	3	65	95	95	95	95	95	95	95
	4	25	80	85	85	85	85	85	85
	Mean	35	80 (+)	86 (+)	86 (+)	88	89	91	91 (-)

(-) / (+) = No statistically / Statistically significant difference from control groups

Deleted values= Replicate not included for calculations. (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

 

 

Swim-up

Table 2: Percent Swim-up of Hatched Live Fry of the Control, Solvent Control and the Test Concentrations of 0.640 to 2.33 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	PHD -2	PHD -1	PHD 0	PHD 1
		Study day 3	Study day 4	Study day 5	Study day 6
		Cumulative swim up [%]
Control	1	0	0	100	100
	2	0	0	100	100
	3	0	0	100	100
	4	0	0	100	100
	Mean	0	0	100	100
Solvent control	1	0	10	100	100
	2	0	0	100	100
	3	0	0	100	100
	4	0	0	100	100
	Mean	0	3	100	100
0.640	1	0	0	100	100
	2	0	0	100	100
	3	0	0	100	100
	4	0	0	100	100
	Mean	0	0	100	100
1.29	1	0	10	100	100
	2	0	0	100	100
	3	0	0	100	100
	4	0	5	100	100
	Mean	0	4	100	100
2.33	1	0	0	100	100
	2	0	0	89	100
	3	0	0	100	100
	4	0	0	100	100
	Mean	0	0	97	100

Table 3: Percent Swim-up of Hatched Live Fry of the Test Concentrations of 4.60 and 9.61 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	PHD -2	PHD -1	PHD 0	PHD 1
		Study day 3	Study day 4	Study day 5	Study day 6
		Swim up [%]
4.60	1	0	11	95	100
	2	0	0	100	100
	3	0	0	100	100
	4	0	0	100	100
	Mean	0	3	99	100
9.61	1	0	0	83	n.a.
	2	0	0	67	n.a.
	3	0	0	29	n.a.
	4	0	0	55	n.a.
	Mean	0	0	59	n.a.

n.a. = not applicable, since abnormal behavioural effects (e.g. quiescence) interferred with swim-up

Deleted values= Replicate not included for calculations (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

 

Fry Survival (Post-Hatch Survival)

Table 4: Post-Hatch Survival on Study Day35 (PHD 30) of the Control, Solvent Control and the Test Item Concentrations of 0.640 to 9.61 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	Eggs introduced on study day 0	Cumulative number of hatched larvae	Vital Larvae on study day 35 (PHD 30)	Post-Hatch survival on study day 35 [%]	Post-Hatch mortality on study day 35 [%]
Control	1	20	20	18	90	10
	2	20	18	13	72	28
	3	20	20	11	55	45
	4	20	20	17	85	15
	Mean	20	19.5	14.8	75.5	25
Solvent control	1	20	20	16	80	20
	2	20	20	16	80	20
	3	20	19	17	89	11
	4	20	18	16	89	11
	Mean	20	19.3	16.3	84.5	16
0.640	1	20	19	17	89	11
	2	20	20	16	80	20
	3	20	19	12	63	37
	4	20	20	15	75	25
	Mean	20	19.5	15.0	77 (-)	23
1.29	1	20	19	15	79	21
	2	20	19	13	68	32
	3	20	17	13	76	24
	4	20	19	15	79	21
	Mean	20	18.5	14.0	76 (-)	24
2.33	1	20	19	13	68	32
	2	20	20	17	85	15
	3	20	20	18	90	10
	4	20	19	15	79	21
	Mean	20	19.5	15.8	81 (-)	19
4.60	1	20	20	12	60	40
	2	20	20	6	30	70
	3	20	17	3	18	82
	4	20	20	9	45	55
	Mean	20	19.3	7.5	31 (+)	69
9.61	1	20	18	3	17	83
	2	20	19	0	0	100
	3	20	19	0	0	100
	4	20	17	0	0	100
	Mean	20	18.3	0.8	4 (+)	96

(-) / (+) = No statistically / Statistically significant difference from control groups

Deleted values= Replicate not included for calculations. (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

 

 

 

Overall Survival (Overall Mortality)

 Table 5: Overall Survival and Overall Mortality on Study Day 35 (PHD 30)of the Control, Solvent Control and the Test Item Concentrations 0.640 and to 1.29 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	Vital larvae on study day 35 (PHD 30)	Overall survival [%]	Overall mortality [%]
Control	1	18	90	10
	2	13	65	35
	3	11	55	45
	4	17	85	15
	Mean	14.8	74	26
Solvent control	1	16	80	20
	2	16	80	20
	3	17	85	15
	4	16	80	20
	Mean	16.3	81	19
0.640	1	17	85	15
	2	16	80	20
	3	12	60	40
	4	15	75	25
	Mean	15.0	75 (-)	25
1.29	1	15	75	25
	2	13	65	35
	3	13	65	35
	4	15	75	25
	Mean	14.0	70 (-)	30

(-) / (+) = No statistically / Statistically significant difference from control groups

 

Table 6: Overall Survival and Overall Mortality on Study Day 35 (PHD 30) of Test Item Concentrations 2.33 to 9.61 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	Vital larvae on study day 35 (PHD 30)	Overall survival [%]	Overall mortality [%]
2.33	1	13	65	35
	2	17	85	15
	3	18	90	10
	4	15	75	25
	Mean	15.8	79 (-)	21
4.60	1	12	60	40
	2	6	30	70
	3	3	15	85
	4	9	45	55
	Mean	7.5	30 (+)	70
9.61	1	3	15	85
	2	0	0	100
	3	0	0	100
	4	0	0	100
	Mean	0.8	5 (+)	95

(-) / (+) = No statistically / Statistically significant difference from control groups

Deleted values= Replicate not included for calculations. (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

 

Morphological and behavioural effects

Table: 7: Behavioural Effects of the Test Concentrations 4.60 and 9.61 mg/L (Study Day 6 – 22)

Study Day	Time-weighted arithmetic mean measured test item concentration [mg/L]
	4.60				9.61
	1	2	3	4	1	2	3	4
6	-	-	-	-	4: G, Q	4: G, Q	1: Q, S 1:T	2:Q
7	2: T	1: T	3: Q	-	-	-	1: L	2: L
8	2: T	1*: Q, G	1: Q	-	1: G, Q	-	-	2: L
9	-	1*: Q, G	-	-	-	1: L	-	2: L
10	-	1*: Q, G	-	-	1: G, Q	1: T	-	2: L
11	-	1: Q, G	-	-	1: G, Q	-	-	1: L
12	-	1: Q, G	-	-	1: G, Q	-	-	1: L
13	-	-	-	-	-	-	-	1: L
14	-	-	-	-	-	-	-	-
15	-	-	-	-	-	-	-	-
16	-	-	1: Q	-	-	-	1: T	-
17	-	1: S	-	-	-	-	-	-
18	-	-	-	-	-	2: Q, G	-	-
19	-	-	-	-	-	1: Q, G	-	-
20	-	-	-	-	-	1: Q, G	-	-
21	-	-	-	-	-	-	-	-
22	-	1: Q, G	-	-	-	-	-	-

* Larvae with scoliosis

 

Table 8: Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30)of the Control, Solvent Control and the Test Item Concentrations of 0.640 to 2.33 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	PHD 30 (End of exposure)
		Mean total length per fish [mm]	Mean wet weight per fish [mg]
Control	1	20.6	71.7
	2	21.3	82.0
	3	20.6	75.4
	4	20.2	72.3
	Mean	20.7	75.4
	± SD	0.457	4.72
	CV [%]	2.21	6.26
Solvent control	1	19.9	67.1
	2	20.1	64.8
	3	19.5	61.4
	4	20.4	67.9
	Mean	20.0	65.3
	± SD	0.378	2.91
	CV [%]	1.89	4.46
0.640	1	19.9	64.0
	2	20.6	71.9
	3	20.1	76.3
	4	19.6	65.8
	Mean	20.1 (-)	69.5 (-)
	± SD	0.420	5.66
	CV [%]	2.09	8.14
1.29	1	20.2	66.1
	2	19.1	68.1
	3	20.5	81.3
	4	18.9	62.0
	Mean	19.7 (-)	69.4 (-)
	± SD	0.793	8.35
	CV [%]	4.03	12.0
2.33	1	18.3	56.2
	2	18.9	54.2
	3	17.2	45.3
	4	18.7	57.8
	Mean	18.3 (+)	53.4 (+)
	± SD	0.759	5.58
	CV [%]	4.15	10.4

(-) / (+) = No statistically / Statistically significant difference from control groups

 

Table 9: Overview of Fry Growth: Length and Wet Weight on Study Day 35 (PHD 30) of the Test Item Concentrations of 4.60 and 9.61 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Rep.	PHD 30 (End of exposure)
		Mean total length per fish [mm]	Mean wet weight per fish [mg]
4.60	1	18.4	54.9
	2	16.3	46.9
	3	16.0	40.7
	4	12.2	20.5
	Mean	14.8 (-*)	36.0 (+)
	± SD	2.29	13.8
	CV [%]	15.4	38.3
9.61	1	9.0	5.03
	2	8.0	n.a.
	3	8.0	n.a.
	4	n.a.	n.a.
	Mean	8.33 (-*)	n.a. (+)
	± SD	0.577	n.a.
	CV [%]	6.93	n.a.

(-) / (+) = No statistically / Statistically significant difference from control groups

* = biologically significant

Deleted values= Replicate not included for calculations. (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

n.a.= Not applicable, due to 100% mortality

 

Table10: Individual Length on Study Day 35 (PHD 30)of the Control, the Solvent Control and the Test Item Concentration of 0.640 mg/L

Fish No.	Time-weighted arithmetic mean test item conc. [mg/L]
	Control				Solvent control				0.640 mg/L
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4	1	2	3	4
1	21.0	21.5	20.0	22.0	20.0	25.0	21.5	23.0	23.0	21.0	20.5	21.0
2	21.5	18.5	19.0	19.5	16.5	21.5	15.5	19.0	24.5	19.0	7.5	18.0
3	21.0	19.0	20.0	18.0	20.0	22.0	22.0	22.0	22.0	24.0	21.5	14.5
4	20.5	22.0	19.5	21.5	23.0	18.5	20.0	20.0	19.0	21.5	20.0	20.0
5	20.5	25.0	20.0	18.0	19.0	19.0	23.5	19.0	18.0	25.0	22.5	23.0
6	17.5	18.5	19.5	14.0	23.5	20.5	23.0	18.5	19.5	18.5	18.5	14.5
7	18.5	25.0	22.0	23.0	22.0	19.5	20.0	22.0	20.0	20.0	21.5	22.0
8	20.0	21.5	23.0	22.0	18.0	22.0	18.0	21.0	19.5	11.5	19.0	22.5
9	21.5	22.0	23.0	20.0	18.0	13.0	18.0	20.5	17.0	20.5	24.0	21.5
10	23.0	18.5	21.5	20.0	21.5	20.5	23.0	20.5	21.0	21.5	21.0	18.0
11	24.0	20.0	19.5	24.5	22.0	18.5	20.5	20.5	19.0	23.5	23.0	23.5
12	21.5	23.0	-	21.0	21.5	22.5	19.0	21.0	18.0	21.5	22.0	18.5
13	17.0	23.0	-	20.0	23.5	19.0	10.5	22.0	19.0	18.0	-	22.0
14	21.5	-	-	20.0	20.5	17.0	20.0	17.0	19.5	20.0	-	20.0
15	22.5	-	-	22.0	17.5	23.0	17.0	20.0	18.0	20.0	-	15.0
16	19.5	-	-	18.5	11.5	19.5	19.5	20.5	20.0	23.5	-	-
17	20.5	-	-	20.0	-	-	20.0	-	20.5	-	-	-
18	19.5	-	-	-	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-
Mean	20.6	21.3	20.6	20.2	19.9	20.1	19.5	20.4	19.9	20.6	20.1	19.6
± SD	1.79	2.32	1.47	2.37	3.12	2.78	3.17	1.52	1.92	3.14	4.27	3.07
CV %	8.69	10.9	7.14	11.7	15.7	13.8	16.3	7.45	9.65	15.2	21.2	15.7

-     = Fish died before end of the study

 

Table 11: Individual Length on Study Day 35 (PHD 30) of Test Item Concentrations of 1.29 to 4.60 mg/L

Fish No.	Time-weighted arithmetic mean test item conc. [mg/L]
	1.29				2.33				4.60
	Total length of individual fish in [mm]
	1	2	3	4	1	2	3	4	1	2	3	4
1	20.0	24.0	20.5	20.0	20.5	20.5	15.0	20.0	17.5	21.0	11.0	12.0
2	25.0	13.0	23.0	22.5	19.5	18.0	20.0	11.5	18.0	13.0	16.0	8.0
3	22.5	24.0	24.5	14.5	20.0	15.5	8.0	20.5	17.0	17.0	21.0	11.0
4	21.0	22.0	18.0	9.0	8.0	23.0	11.0	22.0	15.0	12.0	-	20.5
5	20.0	24.5	20.0	17.0	10.0	17.0	24.5	14.0	15.0	16.5	-	14.5
6	18.0	10.5	19.5	16.0	20.5	19.5	15.5	20.0	23.0	18.0	-	12.5
7	21.0	13.0	11.0	23.0	21.0	20.5	20.0	19.5	20.0	-	-	14.5
8	22.5	19.5	14.0	24.0	22.5	20.0	22.0	21.5	19.0	-	-	11.0
9	18.0	20.0	24.0	20.0	20.0	19.0	20.0	19.5	19.5	-	-	6.0
10	18.0	23.5	22.0	17.0	16.0	15.0	18.0	19.0	21.0	-	-	-
11	21.0	17.5	23.0	20.5	21.0	21.0	17.0	15.0	16.0	-	-	-
12	18.0	17.0	27.0	20.0	20.5	19.5	16.5	16.0	20.0	-	-	-
13	21.5	19.5	20.0	21.5	18.0	18.0	18.5	22.0	-	-	-	-
14	18.0	-	-	19.0	-	20.5	21.0	20.5	-	-	-	-
15	18.0	-	-	19.0	-	20.0	17.5	20.0	-	-	-	-
16	-	-	-	-	-	17.0	20.5	-	-	-	-	-
17	-	-	-	-	-	18.0	13.0	-	-	-	-	-
18	-	-	-	-	-	-	11.0	-	-	-	-	-
19	-	-	-	-	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-
Mean	20.2	19.1	20.5	18.9	18.3	18.9	17.2	18.7	18.4	16.3	16.0	12.2
± SD	2.18	4.67	4.33	3.80	4.42	2.07	4.30	3.13	2.46	3.31	5.00	4.16
CV %	10.9	24.6	21.0	20.1	24.0	11.1	25.0	16.6	13.6	20.2	31.3	34.4

-     = Fish died before end of the study

Deleted values= Replicate not included for calculations. (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

 

Table12: Individual Length on Study Day 35 (PHD 30) of Test Item Concentration of 9.61 mg/L

Fish No.	Time-weighted arithmetic mean test item conc. [mg/L]
	9.61
	Total length of individual fish in [mm]
	1	2	3	4
1	9.0	-	-	-
2	8.0	-	-	-
3	8.0	-	-	-
4	-	-	-	-
5	-	-	-	-
6	-	-	-	-
7	-	-	-	-
8	-	-	-	-
9	-	-	-	-
10	-	-	-	-
11	-	-	-	-
12	-	-	-	-
13	-	-	-	-
14	-	-	-	-
15	-	-	-	-
16	-	-	-	-
17	-	-	-	-
18	-	-	-	-
19	-	-	-	-
20	-	-	-	-
Mean	8.33	n.a.	n.a.	n.a.
± SD	0.577	n.a.	n.a.	n.a.
CV %	6.93	n.a.	n.a.	n.a.

-     = Fish died before end of the study

n.a.= Not applicable, due to 100% mortality

 

Table 13: Pooled Wet Weights on Study Day 35 (PHD 30)of the Control, Solvent Control and the Test Item Concentrations of 0.640 to 9.61 mg/L

Time-weighted arithmetic mean test item conc. [mg/L]	Replicate	Number of fish alive on study day 35	Pooled wet weight per replicate [mg]	Mean wet weight per fish [mg]	Mean [mg]	± SD	CV %
Control	1	18	1290.6	71.7	75.4	4.72	6.26
	2	13	1066.3	82.0
	3	11	829.1	75.4
	4	17	1229.3	72.3
Solvent control	1	16	1073.2	67.1	65.3	2.91	4.46
	2	16	1036.0	64.8
	3	17	1043.7	61.4
	4	16	1085.8	67.9
0.640	1	17	1088.8	64.0	69.5	5.66	8.14
	2	16	1149.9	71.9
	3	12	915.0	76.3
	4	15	987.0	65.8
1.29	1	15	991.5	66.1	69.4	8.35	12.0
	2	13	885.7	68.1
	3	13	1056.6	81.3
	4	15	929.4	62.0
2.33	1	13	731.2	56.2	53.4	5.58	10.4
	2	17	921.8	54.2
	3	18	815.0	45.3
	4	15	867.1	57.8
4.60	1	12	658.6	54.9	36.0	13.8	38.3
	2	6	281.2	46.9
	3	3	122.2	40.7
	4	9	184.2	20.5
9.61	1	3	15.1	5.03	n.a.	n.a.	n.a.
	2	0	n.a.	n.a.
	3	0	n.a.	n.a.
	4	0	n.a.	n.a.

n.a.= Not applicable, due to 100% mortality

Deleted values= Replicate not included for calculations. (Since an application malfunction of the stock solution to the mixing chamber of replicate 1 of the nominal test concentration of 5.00 mg/L was detected on study day 22, the concerning replicate was not included in any evaluation and statistical analysis.)

Validity criteria fulfilled:

yes

Conclusions:

The most sensitive endpoint in a 35-d (30-d post-hatch) FELS toxicity test exposing Danio rerio towards 2-ethylheyl-S-lactate was fry growth expressed as weight with an EC10 of 1.86 mg/L (Time Weighted Mean of measured concentrations, TWM).
The test was conducted according to OECD test guideline 210.

Executive summary:

The 35-day (30 days post-hatch) chronic toxicity of 2-ethylhexyl-S-lactate to early life stage of zebrafish (Danio rerio) was studied under flow through conditions. Fertilized eggs (80 eggs per test concentration, solvent control and control, in total 560 eggs for the main test; stadium: more than 2 cells, less than 24 h old) of D. rerio were exposed to control, solvent control (methanol), and 2-ethylhexyl-S-lactate.The solvent concentration was the same in each test concentration and the solvent control (0.05 mL/L). Nominal test item concentrations of 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/Lwere applied. Since the measured concentrations were slightly below ± 20 % of the nominal concentration, the time-weighted arithmetic mean measured concentrations were calculated: 0.640 – 1.29 – 2.33 – 4.60 – 9.61 mg/L. The test system was maintained at 25.3 to 26.6 ºC and a pH of 7.60 to 8.28. For the parameter hatch no significant effect was found: the NOEC was ≥ 9.61 mg/L. Therefore, the LOEC for hatch was determined to be > above the highest concentration tested (i.e. > 9.61 mg/L).

For the parameters post hatch survival and overall survival (mortality) the NOEC was 2.33 mg/L. Therefore, the LOEC for these parameters was determined to be 4.60 mg/L. The corresponding EC50 value was 4.34. mg/L for post hatch survival and 4.47 mg/L for overall survival. Corresponding EC10 values could not be determined

No significant effect was observed on the endpoint hatch.

The most sensitive endpoint was fry growth (expressed as length and fresh weight) the NOEC was 1.29 mg/L. Therefore, the LOEC for this parameter was determined to be 2.33 mg/L. The corresponding EC10 values for length and weight were determined with 2.57 and 1.86 mg/L. The EC50 values were calculated as 8.04 and 4.53 mg/L.

The sublethal effects included were the cumulative number of hatched larvae, abnormalities, e.g. quiescence, hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence and atypical feeding behaviour, and fish size and weight.

This toxicity study is classified as acceptable and satisfies the guideline requirement for early life toxicity study with fish.

 

Results synopsis

Test organism size/age: Start with fertilized eggs at least 2-cell stadium, less than 24 h old;

Test type: Flow-through

 

Hatch

NOEC ≥ 9.610 mg/L

LOEC > 9.610 mg/L

EC10 (95 % C.I.) > 9.610 (not determinable) mg/L

EC20 (95 % C.I.) > 9.610 (not determinable) mg/L

EC50 (95 % C.I.) > 9.610 (not determinable) mg/L

 

Length (fry growth)

NOEC = 1.29 mg/L

LOEC = 2.33 mg/L

EC10 (95 % C.I.) = 2.57 (1.94–3.41) mg/L

EC20 (95 % C.I.) = 3.80 (2.88–5.03) mg/L

EC50 (95 % C.I.) = 8.04 (5.38–11.8) mg/L

 

Weight (fry growth) - most sensitive endpoint

NOEC = 1.29 mg/L

LOEC = 2.33 mg/L

EC10 (95 % C.I.) = 1.86 (1.15–3.00) mg/L --> value carried forward for risk assessment

EC20 (95 % C.I.) = 2.53 (1.61–4.01) mg/L

EC50 (95 % C.I.) = 4.53 (2.50–8.07) mg/L

 

Post hatch survival

NOEC = 2.33 mg/L

LOEC = 4.60 mg/L

LC10 (95 % C.I.) = Not determinable

LC20 (95 % C.I.) = Not determinable

LC50 (95 % C.I.) = 4.34 (2.46–7.84) mg/L

 

Overall survival

NOEC = 2.33 mg/L

LOEC = 4.60 mg/L

LC10 (95 % C.I.) = Not determinable

LC20 (95 % C.I.) = Not determinable

LC50 (95 % C.I.) = 4.47 (2.38–8.55) mg/L

Description of key information

In a fish, early-life stage toxicity test according to OECD test guideline 210 and conducted under GLP, fertilized eggs of Danio rerio were exposed towards 2-ethylheyl-S-lactate for 35 d (30 d post-hatch).

The most sensitive endpoint was fry growth expressed as weight resulting in an EC₁₀ of 1.86 mg/L (time weighted mean of measured concentrations, TWM).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.86 mg/L