Benzyl benzoate

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:

2021-01-19 - 2021-03-04

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sample volume per sample was 20 mL. Samples were taken in weekly intervals. Samples were stored protected from light in glass bottles of appropriate volume (e.g. bottle volume at least twice the sample volume) at ≤–18 °C.


Sampling procedure: The samples were taken followed by the procedure described in the following.
- The fish were fed ad libitum early in the morning with one portion of protozoa and/or fry food;
- The test vessels were cleaned 30–60 min after feeding, and mostly all remaining food and faeces were removed from the test vessels (during cleaning the flow-through device was not stopped delivering freshly prepared test solution);
- The new stock solution was prepared and connected to the flow-through device, shortly after cleaning or in parallel to the cleaning procedure;
- After all test vessels are cleaned, the flow-through was continued delivering freshly prepared stock solution (test solutions) into the test vessels;
- The analytical samples were taken after preparation and delivering of at least half of the nominal test vessel volume of freshly prepared test solutions into the test vessels (approximately 400 mL of freshly prepared test solutions, 5–6 preparation cycles (runs) of the flow-through system).

Immediately after sampling and until transfer to the analytical test site, the samples were stored in a freezer (e.g. at –18 °C) in amber glass bottles. The sample volume for analysis was 10 mL for the stock solution (S1) and 20 mL for the stock solution (S2) and the test solution samples. In addition, reserve samples were taken and supplemented with 10% toluene before freezing.

Analyses of the stock solution (S2) were made on samples of the corresponding solutions - when freshly prepared and before renewal.

Vehicle:

no

Details on test solutions:

Preparation of Stock Solution
One stock solution (S1) at a nominal concentration of 10.0 mg test item/L was prepared every day by dissolving the test item in modified reconstituted water in a 5 L measuring flask. This solution was intensively stirred on a magnetic stirrer (1100 rpm) at room temperature for at least 15 minutes interrupted by several short treatments (< 1min) of the solution using an ultrasonic bath. The solution was visually checked for small test item droplets within the water phase, on the water surface and at the bottom of the preparation flask, and the absence of droplets was recorded. If still undissolved test item was observed after this procedure, the procedure was continued until all test item was dissolved. After the test item was completely dissolved, this stock solution S1 was used to prepare the second stock solution (S2) at a nominal concentration of 2.50 mg test item/L. Therefore, the required amount of S1 (5 L) was diluted with modified reconstituted water (15 L). The emptied preparation flask of the S1 was subsequently filled with modified reconstituted water and the modified reconstituted water of the refiled measuring flask was used to prepare the S2 in a stainless steel tank, followed by additional 10 L of modified reconstituted water was added using a 10 L measuring flask. The final stock solution (S2) was stirred for at least 0.5 minutes using a stainless-steel propeller stirrer. This stock solution (S2) was diluted with modified reconstituted water to prepare the desired test concentrations using an automatic flow-through system. The stock solution (S2) was used to produce the desired test item concentration for about one day. The stock solution (S2) was stored at room temperature in the dark while in use.

Preparation of Test Solutions
The test solutions were prepared by a flow-through device. The stock solution S2 and the dilution medium (test medium) were delivered by the flow-through system to mixing vessels. The final test solutions were delivered directly from the mixing vessels to the designated test vessels using teflon tubing and a multi-channel peristaltic pump assembled with Tygone® tubes, set to deliver the desired volume.

During dosing into the flow-through system (between renewals), the control and the stock solution were stored at room temperature in the dark. During storage, the tanks were closed with a lid to reduce air-exchange and evaporation.
The flow rates (volume delivered per stroke) were measured volumetrically at least three times per week. The volume delivered/measured was divided by the number of strokes. The variation of the nominal stroke-volume was calculated by dividing the measured flow [mL/stroke] by the nominal volume per stroke of the designated pump expressed in percentage of nominal values.
The actual measured flow rates for all treatments were within ±10% of the mean flow rates during the exposure period, except at the lowest concentration level (0.050 mg test item/L) at day 18 at which the flow rate of the application solution was slightly lower (89.4% of nominal).
Note (conditioning phase): The flow-through system and the test vessels were conditioned with the test solutions for two day prior to start of exposure. The actually measured flow rates for all treatments during the conditioning phase were within ±10% of the mean flow rates.
The test vessels containing 800 mL of the test solution were placed into a temperature-controlled water bath at test temperature for temperature adaptation prior to test start.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Danio rerio Hamilton-Buchanan 1822 (Zebrafish).

Origin: Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since February 2019.

Holding conditions and fertilisation of test organisms: In the culture tanks, the fish were kept in reconstituted water (OECD guideline 203, 1992) diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater (Tropic Marin, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28 ± 2‰). Fish were held in glass aquaria at 26 ± 2 °C with a 16 h/8 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. (Sanders Brine Shrimp Co., Morgan, UT 84050, U.S.A.) and TetraMin (Tetra Werke, Melle, Germany). A record of holding and health conditions is kept.

Exposure (Collecting of Eggs):
On the evening before test start, glass bowls covered with stainless steel mesh were introduced in the holding aquaria of the adult zebrafish. Water plants were placed on the mesh, allowing the fish to spawn.

The spawning bowls were removed from the holding aquaria on the day of test start shortly after onset of illumination (i.e. 45 minutes), which triggers fertilization. The content of the bowls was poured over a sieve, rinsed with tempered reconstituted water and collected in a glass vessel filled with tempered reconstituted water. Immediately afterwards, groups of eggs were transferred to glass dishes (one replicate per test concentration) containing test solution of each designated exposure vessel, acclimated at test temperature. After each pre-exposure vessel contained about 100 eggs, the dishes were placed into an incubator set to 26 °C (26 ± 1.5 °C) for 2 hours. Thereafter, groups of fertilised eggs were transferred to an additional set of pre-exposure vessels (four replicates per test concentration) containing 50 mL of temperature-adapted test solutions using a randomisation procedure. This was done by sequentially adding groups of fertilised eggs (5 eggs per test vessel) from the first pre-exposure vessel for each test concentration into the corresponding replicated pre-exposure vessels until each test vessel contained 20 eggs. Thereafter the fertilised eggs (20 per pre-exposure vessel) were transferred into the test units.

Test type:

flow-through

Water media type:

freshwater

Remarks:

five vessel volumes per vessel and day from day 0 – 14; increased to ten vessel volumes per vessel and day from day 14 (10:15h) – 35 to assure stable test item concentrations during a test period of high food ratio and higher loading rate of fish larvae.

Limit test:

no

Total exposure duration:

35 d

Remarks on exposure duration:

Newly fertilised eggs were exposed to the test item dissolved in test medium in a flow-through system for 30 days post-hatch.

Hardness:

Total water hardness was measured in each test vessel at the start and the end of the exposure period.
Range: 164 - 175 mg CaCO3/L; 9.6 - 9.8 °dH

Test temperature:

The water temperature should be within a range of ±1.5 °C of the desired test temperature (26°C).
Temperatures observed were:
Min.: 26.3 °C, Max.: 26.7 °C (automatic measurement)
Min.: 25.7 °C, Max.: 26.7 °C (manual measurement)

pH:

pH was measured in each test vessel at the start and the end of the exposure period.
Range: 7.4 - 7.6

Dissolved oxygen:

Minimum: 77%
Range: 7.5 - 8.4 mg/L

Salinity:

not applicable

Conductivity:

Range: 752 - 799 µS/cm

Nominal and measured concentrations:

Test concentrations: 0.050, 0.089, 0.158, 0.281 and 0.500 mg test item/L (nominal) + Control were used.

During the exposure period the concentration in the test solutions showed by trend lower test concentration than determined at start of the exposure period. At the three highest treatment levels (0.158 to 0.500 mg test item/L) the recoveries ranged between n.a. (
The resulting geometric mean measured concentrations were 0.0056, 0.0125, 0.0228, 0.0486 and 0.190 mg test item/L, equivalent to mean recoveries of 11.2, 14.0, 14.4, 17.3 and 38.0% of nominal concentrations.

Details on test conditions:

Test Units

Glass vessels of appropriate sizes (1000 mL volume) were used as test vessels.

• Dimensions of test vessels:
o Diameter: 14.0 cm, Height: 8 cm; vessels were fitted with a sideward fixed meshed overflow.
• Volume of test solution per test vessel: 0.8 L (nominal).
• Test vessels covered with a transparent cover.

xposure Conditions

• Photoperiod: light/dark cycle 14 h/10 h.
• Light intensity: measured: 605 – 888 lx.
• Temperature: 26 ± 1.5 °C (target); measured min/max: 25.7/26.7 °C (manual); 26.3/26.7°C (automatic).
• Aeration: the test vessels were gently aerated during the test using teflon tubes, and were controlled daily.
• Renewal of the test solution during the test period: flow-through, five vessel volumes per vessel and day from day 0 – 14 and was increased to ten vessel volumes per vessel and day from day 14 (10:15h) – 35 to assure stable test item concentrations during a test period of high food ratio and higher loading rate of fish larvae.
• The test vessels were set up in a quasi-stochastic manner.
• Each test vessel was labelled with the study number, the concentration or concentration code and specific letters for each replicate; each test vessel was closed by a lid.
• The flow rate was adjusted so that the loading (biomass per volume of test solution) did not exceed a loading of 5 g fish/L and a loading rate of 0.5 g fish/L per 24 hours at any time of the test (see below).
• To calculate the maximum loading, the maximum total fish wet weight per vessel (0.6735 g in the controls, replicate b) was divided by the vessel volume (0.8 L). The maximum loading was 0.84 g fish/L.
• The loading rate was calculated by dividing the maximum total fish wet weight per vessel (0.6735 g in the controls, replicate b) by the target flow rate of five vessel volumes per day (4.0 L/vessel/day), respectively ten vessel volumes per day (8.0 L/vessel/day). The maximum loading rate was 0.168 g fish/L per 24 hours, respectively 0.084 g fish/L per 24 hours.
• Feeding: On day 3 of exposure, one day after the first larva per test vessel was recorded to be hatched, feeding was started by adding food to the vessels. The test organisms were fed on dry food (Novo Baby 01 and Baby 02, JBL GmbH & Co. KG, Neuhofen, Germany), live Artemia nauplius larvae and paramaecia (Paramecium caudatum) ad libitum. The daily ration was fed in 2 – 4 equal portions each day, whereas the daily ratio was reduced to a maximum of 3 equal portions per day from day 14 of exposure. The food ration was adjusted to the number of living fish per test vessel. Food was withheld from the fish for 24 hours prior to test end.
• Uneaten food and faeces were removed twice daily on workdays, preferably in the morning, and prior the last feeding in the afternoon, with one exception on day 20 of the exposure period. On weekends the cleaning procedure was partly reduced, and uneaten food was removed once daily.
• Dead eggs, embryos or fish were removed from the test vessels upon daily observation throughout the test.

Reference substance (positive control):

not required

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post-hatch survival

Key result

Duration:

35 d

Dose descriptor:

EC10

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: number of healthy fish

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: number of healthy fish

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: number of healthy fish

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

>= 0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

> 0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight and dry weight, as well as lenghth of surviving fish

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

>= 0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight and dry weight, as well as lenghth of surviving fish

Details on results:

The concentration of the test item in the stock solution showed stable concentration at comparable levels throughout the test (within ±20% of nominal concentration). Nevertheless, the measured concentrations in the test solutions at start of the exposure period (day 0) drop down to recoveries of <60% of nominal concentrations. During the exposure period the concentration in the test solutions showed by trend lower test concentration than determined at start of the exposure period. At the three highest treatment levels (0.158 to 0.500 mg test item/L) the recoveries ranged between n.a. (
Note: This reduction seems to be not clearly corelated with the time duration after adding of food to the test system and the growth of the test organisms, and potential adsorption/degradation processes taking place by present of organic material /microbial activity in the test vessels. Nevertheless, no evidence is available to constrain these suppositions of different degradation pathways of the test item in this test system.

To calculate the measured concentrations of the test item at each treatment level, the geometric mean concentrations of the concentration levels were calculated. Measured concentrations of the test item determined to be
The resulting geometric mean measured concentrations were 0.0056, 0.0125, 0.0228, 0.0486 and 0.190 mg test item/L, equivalent to mean recoveries of 11.2, 14.0, 14.4, 17.3 and 38.0% of nominal concentrations.

The biological data were reported based on nominal test item concentrations in mg test item/L, and based on geometric mean measured test item concentrations in mg test item/L.

Reported statistics and error estimates:

To calculate effect concentrations (ECx-values) different statistical methods were used, i.e. 3-parametric cumulative distribution function (CDF), Weibull and Probit analysis.

The normal distribution was checked with Shapiro-Wilk's Test (wet/dry weight of fish and length of the surviving fish).

Variance homogeneity was checked with Levene´s Test (wet/dry weight of fish and length of the surviving fish).

The trend of the biological data was checked using a trend analysis by contrasts (monotonicity of concentration response); (hatching success, post-hatch survival, wet/dry weight of fish and length of the surviving fish).

To calculate threshold levels (NOEC/LOEC) different statistical methods were performed using Williams Multiple Sequential t-test procedure (wet/dry weight of fish and length of the surviving fish), and Chi2-2x2 table test with Bonferroni correction (hatchability, post-hatch survival).

The statistical software package ToxRat 3.3.0 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Twenty eggs per replicate were exposed. The fish started hatching on day 2 after start of exposure, whereas the hatch of fish larvae started earlier in the higher concentration levels, especially at the highest concentration level (0.500 mg test item/L). Hatching was finished on day 5 after start of exposure for all treatments. The mean hatching success was 93.7% in the control, and between 86.3% and 95.0% in the treated concentration levels.

Overview Hatchability
Cumulative numbers (n) of introduced eggs, hatched fish and unhatched fish (coagulated eggs) and percentage of hatched fish (hatching success) in the control and the treatments (values of four pooled replicates per treatment).

Nominal conc.	Measured conc.	Introduced eggs	Hatched fish	Not Hatched	Not Hatched	Hatching success
[mg test item /L]		[n]			[%]
Control	Control	80	75	5	6.3	93.7
0.050	0.0056	80	69	11	13.7	86.3
0.089	0.0125	80	74	6	7.5	92.5
0.158	0.0228	80	76	4	5.0	95.0
0.281	0.0486	80	71	9	11.3	88.7
0.500	0.190	80	75	5	6.3	93.7

The NOEC based on nominal concentration was ≥0.500 mg test item/L; equivalent >0.190 mg test item/L based on measured concentrations.

No correlation was observed between the concentration of the test item and the hatching success of the fish larvae. Therefore, ECx values were not calculated for the parameter hatching success of fish larvae at the end of the exposure period. The EC50 was determined to be >0.500 mg test item/L based on nominal concentrations; equivalent to >0.190 mg test item/L based on measured concentrations.

Overview Post-hatch survival

Cumulative numbers (n) of hatched, surviving and dead fish larvae, and percentage of dead fish larvae (mortality of hatched fish, post-hatch mortality) and survival of hatched fish larvae (post-hatch success) assessed at test end in the control and treatments.

Nominal concentration	Measured concentration	Hatched fish larvae	Surviving fish larvae	Dead fish larvae	Post-hatch mortality	Post-hatch success (survival)
[ mg test item / L]		[n]			[%]
Control	Control	75	66	9	12.0	88.0
0.050	0.0056	69	59	10	14.5	85.5
0.089	0.0125	74	69	5	6.8	93.2
0.158	0.0228	76	69	7	9.2	90.8
0.281	0.0486	71	48	23	32.4	67.6
0.500	0.190	75	4	71	94.7	5.3

The NOEC based on nominal concentration was 0.158 mg test item/L, equivalent to 0.0228 mg test item/L based on measured concentrations.

A clear correlation was observed between the concentration of the test item and the d post-hatch success of fish larvae. Therefore, ECx values were calculated for the parameter post-hatch success of fish larvae at the end of the exposure period. The EC50 was determined to be 0.352 mg test item/L based on nominal concentrations, equivalent to 0.0913 mg test item/L based on measured concentrations.

Summary

Summary of the total numbers of pre- and post-hatch survival and sub-lethal data, and mean weights and length data of surviving fish larvae in the control and treatment at test end.

Nominal concentration	Measured concentration	Introduced eggs	Hatched eggs	Survived fish	Deformed fish/ Different behavior	Healthy fish	Wet weight of fish	Dry weight of fish	Length of fish
[mg test item / L]	[mg test item / L]	[n]	[n]	[n]	[n]	[n]	[mg]	[mg]	[mm]
Control	Control	80	75	66	0	66	34.70	9.42	16.70
0.050	0.0056	80	69	59	0	59	38.21	10.95	16.84
0.089	0.0125	80	74	69	0	69	36.02	9.67	16.33
0.158	0.0228	80	76	69	0	69	36.13	9.80	16.49
0.281	0.0486	80	71	48	0	48	39.75	10.85	16.99
0.500	0.190	80	75	4	0	4	35.73	9.78	18.05

Overview Number of healthy fish

Mostly all surviving fish appeared healthy at the end of exposure (day 35), i.e. behavioural or morphological abnormalities were not observed; except at the highest concentration level all surviving fish showed slight behavioural differences compared to the control. Since the number of unhealthy fish at the end of exposure seems to be correlated with the increase of test concentrations, a separate statistical evaluation was performed on the number of healthy fish.

Abnormal behaviour (e.g. lying on the bottom of the vessels) of fish larvae observed during the test (from day 5 – 35 after start of the exposure) follow a concentration-relationship and is therefore considered to be test item related

Cumulative numbers (n) of hatched, surviving, deformed fish (fish showing different behaviour) and healthy fish larvae, and percentage of healthy fish larvae of hatched fish larvae (post-hatch success) assessed at test end in the control and treatments C1 – C5 (values of four pooled replicates per treatment).

Nominal concentration	Measured concentration	Hatched fish larvae	Surviving fish larvae	Deformed fish	Healthy fish	Healthy fish (% of hatched fish)
[ mg test item / L]		[n]			[%]
Control	Control	75	66	0	66	88.0
0.050	0.0056	69	59	1	58	84.1
0.089	0.0125	74	69	0	69	93.2
0.158	0.0228	76	69	1	68	89.5
0.281	0.0486	71	48	0	48	67.6
0.500	0.190	75	4	4	0	0

The NOEC based on nominal concentration was 0.158 mg test item/L, equivalent to 0.0228 mg test item/L based on measured concentrations.

A clear correlation was observed between the concentration of the test item and the numbers of healthy fish larvae at the end of the exposure period. Therefore, ECx values were calculated for the parameter post-hatch success of fish larvae at the end of the exposure period. The EC10 was determined to be 0.207 mg test item/L based on nominal concentrations, equivalent to 0.0323 mg test item/L based on measured concentrations.

Fresh weight in Dani rerio after 35d

Per-treatment individual mean wet weights in mg at test end (values of pooled replicates per treatment).

Nominal concentration [mg test item/L]	Mean [mg]	Std. Dev. [mg]	Replicate number [n]	Difference compared to control [%]
Control	34.700	3.8971	4
0.050	38.213	6.5032	4	-10.1
0.089	36.023	0.6184	4	-3.8
0.158	36.130	3.1891	4	-4.1
0.281	39.747	0.6881	4	-14.5
0.500	35.725	0.1909	2	-30

In the control the mean wet weight was 34.70 mg. In the treatments the mean wet weight was within a range of 35.73 and 39.75 mg.

An influence of the test item on the wet weight of the surviving fish could not be observed.

Statistically significant differences of wet weight of fish were not observed between treated and control groups at p≤0.05. The NOEC based on nominal concentration was ≥0.500 mg test item/L, equivalent to ≥0.190 mg test item/L based on measured concentrations. No correlation was observed between the concentration of the test item and the wet weight of fish. Therefore, no ECx values were calculated for the parameter fresh weight of fish larvae at the end of the exposure period. The EC10 was determined to be >0.500 mg test item/L based on nominal concentrations, equivalent to >0.190 mg test item/L based on measured concentrations.

Dry weight in Dani rerio after 35d

Per-treatment individual mean dry weights in mg at test end (values of pooled replicates per treatment).

Nominal concentration [mg test item/L]	Mean [mg]	Standard deviation [mg]	Replicate number [n]	Difference compared to control [%]
Control	9.42	1.362	4
0.050	10.95	1.956	4	-16.2
0.089	9.72	0.337	4	-3.1
0.158	9.80	1.229	4	-4.0
0.281	10.81	0.363	4	-14.8
0.500	9.78	0.049	2	-3.8

In the control the mean dry weight was 9.42 mg. In the treatments the mean dry weight was within a range of 9.67 and 10.95 mg. An influence of the test item on the dry weight of the surviving fish could not be observed. Statistically significant differences of dry weight of fish were not observed between treated and control groups at p≤0.05. The NOEC based on nominal concentration was ≥0.500 mg test item/L, equivalent to ≥0.190 mg test item/L based on measured concentrations.

No correlation was observed between the concentration of the test item and the dry weight of fish. Therefore, no ECx values were calculated for the parameter dry weight of fish larvae at the end of the exposure period. The EC10 was determined to be >0.500 mg test item/L based on nominal concentrations, equivalent to >0.190 mg test item/L based on measured concentrations.

Lenghth in Danio rerio after 35d

Per-treatment mean standard lengths in cm at test end (values of pooled replicates per treatment).

Nominal concentration [mg test item/L]	Mean [mm]	Standard deviation [mm]	Replicate number [n]	Reduction compared to control [%]
Control	16.70	0.668	4
0.050	16.84	0.555	4	-0.9
0.089	16.33	0.232	4	2.2
0.158	16.49	0.472	4	1.3
0.281	16.99	0.312	4	-1.8
0.500	18.05	3.182	2	-8.1

In the control the mean length was 16.70 mm at test end. In the treatments the mean fish length within a range of 16.33 and 18.05 mm at test end.

An influence of the test item on the length of the surviving fish could not be observed.

Statistically significant differences of length of the surviving fish were not observed between treated and control groups at p≤0.05. The NOEC based on nominal concentration was ≥0.500 mg test item/L, equivalent to ≥0.190 mg test item/L based on measured concentrations.

No correlation was observed between the concentration of the test item and the length of the surviving fish. Therefore, no ECx values were calculated for the parameter length of the surviving fish larvae at the end of the exposure period. The EC10 was determined to be >0.500 mg test item/L based on nominal concentrations, equivalent to >0.190 mg test item/L based on measured concentrations.

Validity criteria fulfilled:

yes

Conclusions:

The substance was investigated with respect to long-term toxicity towards fish according to OECD Guideline 210. A clear correlation was observed between the concentration of the test item and the d post-hatch success of fish larvae. Therefore, ECx values were calculated for the parameter post-hatch success of fish larvae at the end of the exposure period. The EC50 was determined to be 0.0912 mg test item/L based on measured concentrations. The EC10 was determined to be 0.0342 mg test item/L based on measured concentrations, with NOEC = 0.228 mg test item / L and LOEC = 0.0486 mg test item /L measured concentration, respectively.
Calculation of the number of healthy fish yields slightly different to data for post-hatch success, therefore a separate statistical evaluation was performed. The EC10 value of the number of healthy fish is 0.0322 mg test item / L, based on measured concentrations.

Executive summary:

The substance is investigated with respect to long-term toxicity towards fish according to OECD Guideline for Testing of Chemicals, No. 210 "Fish, Early-life Stage Toxicity Test". Adopted on July 26, 2013. Organisation for Economic Co-Operation and Development, Paris. The aim of the study is to determine lethal and sub-lethal effects of the test item on the early-life stages of Danio rerio (zebrafish).

Eggs/fish of Danio rerio were exposed under control conditions to untreated test medium (mod. reconstituted water) as well to the f

ollowing nominal concentrations of the test material: 0.050, 0.089, 0.158, 0.281 and 0.500 mg test item/L.

Test solutions were continuously renewed in a flow-through system. Four replicates each containing 20 fertilised eggs/fish were used per test concentration and control. Exposure conditions were constantly monitored. To determine the actual test item concentrations, samples were taken from the stock and test solutions for analytical measurement. Samples were measured in stock and test solutions at regular intervals throughout the exposure period. The measured test item concentrations in the freshly prepared and aged stock solutions ranged between 2.03 and 2.59 mg test item/L, corresponding to recoveries between 81.2 and 104% based on nominal concentrations. The overall geometric mean concentration for the stock solutions was 2.32 mg test item/L.

 

During the exposure period the concentration in the test solutions showed by trend lower test concentration than determined at start of the exposure period. At the three highest treatment levels (0.158 to 0.500 mg test item/L) the recoveries ranged between n.a. (<LOD) and 48.2% of nominal concentrations. At the lower concentration levels, 0.050 and 0.089 mg test item/L the measured concentrations were always <LOD/LOQ, and also at some occasions at the concentration levels 0.158 and 0.281 mg test item/L.

 

To calculate the biological endpoints (NOEC/LOEC and ECx) based on measured concentrations, a geometric mean measured concentration was calculated for each concentration level. The resulting geometric mean measured concentrations were 0.0056, 0.0125, 0.0228, 0.0486 and 0.190 mg test item/L, equivalent to mean recoveries of 11.2, 14.0, 14.4, 17.3 and 38.0% of nominal concentrations.

The determined endpoints are summarized as follows:

	Endpoints (measured) [mg test item / L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Hatching Success	n.d. (> 0.190)	n.d. (>0.190)	n.d. (>0.190)	> 0.190	>= 0.190
Post-hatch success (survival)	0.0342	0.0505	0.0912	0.0228	0.0486
Number of healthy fish	0.0322	0.0451	0.0750	0.0228	0.0486
Length of the surviving fish	n.d. (>0.190)	n.d. (>0.190)	n.d. (>0.190)	> 0.190	>=0.190
Wet weight of surviving fish	n.d. (>0.190)	n.d. (>0.190)	n.d. (>0.190)	> 0.190	>= 0.190

 ¹ Data are slightly different with data for post-hatch success, therefore a separate statistical evaluation was performed.

n.d.: not determined due to mathematical reasons and/or inappropriate data

* The given ECx-values were not statistically evaluated, since for all biological parameter no concentration-response relationship was observed, in addition the determined inhibitions were always <16%, therefore the ECx-values were stated to be higher than the highest measured concentration level.

 

• An influence of the test item on the hatching success could not be observed. A NOEC >= 0.190 mg test item/L and LOEC > 0.190 mg test item/L based on measured concentrations was determined


• A clear correlation was observed between the concentration of the test item and the d post-hatch success of fish larvae. Therefore, ECx values were calculated for the parameter post-hatch success of fish larvae at the end of the exposure period. The EC₅₀ was determined to be 0.352 mg test item/L based on nominal concentrations, equivalent to 0.0912 mg test item/L based on measured concentrations. The EC₁₀was determined to be 0.0342 mg test item/L based on measured concentrations, with NOEC = 0.228 mg test item / L and LOEC = 0.0486 mg test item /L measured concentration, respectively.


• Calculation of the number of healthy fish yields slightly different to data for post-hatch success, therefore a separate statistical evaluation was performed. The EC₁₀ value of the number of healthy fish is 0.0322 mg test item / L, based on measured concentrations, with NOEC = 0.228 mg test item / L and LOEC = 0.0486 mg test item /L measured concentration, respectively. .


• No correlation was observed between the concentration of the test item and the wet weight of fish. A NOEC >= 0.190 mg test item/L and LOEC > 0.190 mg test item/L based on measured concentrations was determined.


• Statistically significant differences of dry weight of fish were not observed between treated and control groups. A NOEC >= 0.190 mg test item/L and LOEC > 0.190 mg test item/L based on measured concentrations was determined.


• An influence of the test item on the length of the surviving fish could not be observed. A NOEC >= 0.190 mg test item/L and LOEC > 0.190 mg test item/L based on measured concentrations was determined.

Description of key information

Under the experimental conditions of this study, a clear concentration-response relationship was observed for the parameter post-hatch success (survival) and number of healthy fish. No relationship was observed for the parameter hatching success, length, wet and dry weight of the surviving fish. To calculate the biological endpoints (NOEC/LOEC and ECx) based on measured concentrations, a geometric mean measured concentration was calculated for each concentration level. The resulting geometric mean measured concentrations were 0.0056, 0.0125, 0.0228, 0.0486 and 0.190 mg test item/L, equivalent to mean recoveries of 11.2, 14.0, 14.4, 17.3 and 38.0% of nominal concentrations. Lowest effect values were found for "number of healthy fish". The EC10 was determined to be 0.0322 mg test item/L  based on measured concentrations, with NOEC = 0.228 mg test item / L and LOEC = 0.0486 mg test item /L measured concentration, respectively.

Calculation of the number of healthy fish yields slightly different to data for post-hatch success, therefore a separate statistical evaluation was performed. The EC10 value for "post hatch success" is 0.0342 mg test item / L, based on measured concentrations.

 

As the EC10 value is statistically more robust and therefore recommended for assessment in ECHA Guidance Document R.10, and sinde the EC10 value based on the number of healthy fish is the lower value, this value is conervatively used for assessment and derivation of PNEC aqua.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

0.032 mg/L