2-Ethylhexyl trans-4-methoxycinnamate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 2018-09-18 to 2019-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Analytical monitoring:

yes

Remarks:

LC-MS/MS

Details on sampling:

One sample (20 mL) from the control and the treatments was collected from one replicate weekly with the exception of the last sample set which was collected at test conclusion. Thus, during the course of exposure replicate A, B, C, and D were sampled on study days 0, 7, 14, 21, and test termination in rotation, respectively. In addition, stock solutions were analysed with diluter samples. Samples were centrifuged (2795 x g or 5000 rpm for 30 min) and analysed using LC-MS/MS mehtod.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

- Preparation of saturator columns:
Eight saturator columns were prepared, two for control groups and six for preparation of test item stock solutions. In general, columns were prepared by 125 g/arm pyrolytically cleaned glass wool into each arm of u-shaped glass columns (diameter = 1.54 cm, length = 152.4 cm). To further clean the glass wool, the glass wool was subsequently rinsed with acetone and the acetone purged from the system prior to test substance loading. For the columns used to prepare test substance stock solution, approximately 10 g of the test item was dissolved in 30 mL acetone and distributed equally to both arms of the column. A vacuum pump was used to pull the solution through the glass wool to provide an even coat. The acetone was then evacuated from the columns leaving the test item. Once the saturator columns were dried and the acetone evacuated, the columns were plumbed, and dechlorinated tap water pumped through them at a rate of 4 mL/min for 10 days to rinse the columns and remove any glass particles from the glass wool.
- Determination of the water solubility:
Prior to in-life testing, water solubility was determined at test conditions. On three consecutive days, three samples were collected, centrifuged (2795 x g or 5000 rpm for 30 min.), and preserved. After collection, samples were stored at 4 °C (1 - 9 °C). Once all samples were collected, centrifuged, and preserved, samples were analysed. Two primary factors were used to determine if a successful determination of practical water solubility was achieved: 1) in-life test conditions were maintained throughout assessment, and 2) differences between samples on consecutive days was ≤20 %.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: in-house culture

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Breeding pairs were selected from brood stock of the same age and were maintained together for 8 h following embryo collection. Newly fertilized embryos were selected from a single clutch. All embryos from the clutch selected for use were viewed under a dissecting microscope to determine fertilization and ensure normal development was progressing. Fertilization and viability in the clutch used in the in-life exposure were both ≥90 %. Exposure was initiated prior to 4 hpf (hours post-fertilisation).

POST-HATCH FEEDING
- Type/source of feed: brine shrimp
- Amount given: ad libidum
- Frequency of feeding: twice per day

Test type:

flow-through

Water media type:

freshwater

Remarks:

Dechlorinated (DeCl2) tap water

Limit test:

no

Total exposure duration:

30 d

Remarks on exposure duration:

The in-life exposure duration was 30 dpf (days post fertilisation).

Hardness:

144 - 208 mg/L as CaCO3

Test temperature:

26.2 - 26.7 °C

pH:

7.3 - 7.9

Dissolved oxygen:

6.7 - 7.3 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 2.0 and 20.0 µg/L
Mean Measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 L glass aquaria (with approximate measurements of 40 x 20 x 20 cm)
- Material, size, headspace, fill volume: water-contact components of glass (aquaria), stainless steel (diluter housing and water bath), and Teflon (tubing responsible for test material delivery)
- Aeration: yes, for all test vessels was used to maintain dissolved oxygen levels.
- Type of flow-through: continuous-flow diluter
- Renewal rate of test solution (frequency/flow rate): 2.7 L/h (6.5 volume exchanges/d)
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated (DeCl2) tap water
- Total organic carbon: 0.6 mg/L
- Iodide: 8.8 - 8.9 µg/L
- Ammonia: <0.06 µg/L
- Chlorine: <0.05 µg/L
- Alkalinity: 40 - 108 mg/L as CaCO3
- Intervals of water quality measurement: twice weekly

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 723 - 923 lux

EFFECT PARAMETERS MEASURED: survival, hatch success

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- As a range-finding test, a determination of the water solubility of the test item in the test media was performed. Using recirculating saturator columns over a nine-day period the water solubility of the test item was determined to be 14.7 ± 0.912 μg/L. Based on this results the concentrations for the test were chosen.

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 30

Reference substance (positive control):

no

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

>= 30 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

>= 30 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Other biological observations: normal appearance/behaviour

Confirmation of the test concentrations

Target test item concentrations selected for the FSDT range-finding study (current study) were 0.0 (control), 2.00, and 20.0 μg/L. The dilution water control solutions showed no detectable levels of the test item (<LOQ, <3.04 μg/L). Since time interval between sample collection events was consistent throughout study, the mean measured concentration represented a reasonable estimation of exposure concentration. Because a continuous stock solution was prepared using solid-liquid phase saturator columns, stock concentrations varied slightly during the course of the study. However, impact on test solution concentrations was minimal. The mean measured concentration represented the average of each data point from day 0, 7, 14, 21, and 30 for the sequential sampling of each replicate, A-D, of the control and each treatment. The corresponding mean measured concentrations for the target concentrations of 2.00 and 20.0 μg/L in the range-finding study were 5.84 ± 0.629 and 30.0 ± 1.15 μg/L test item, respectively. Overall, the mean measured concentration was greater than the original target; however, the concentration measured during the in-life exposure was reasonably consistent. The reason for the increase is unclear.

The mean measured concentrations for the 0.0 (control), 2.00, and 20.0 μg/L test item treatment initial targets were <0.912 μg/L [LOD], 5.84 ± 0.629, and 30.0 ± 1.15 μg/L test item.

Table 1. Summary of mean measured concentrations

Test Day	Target Concentration (µg/L)^1	Mean Measured Concentration (µg/L)²	SEM³ (%)
Mean Measured Concentrations	0.0	<LOQ*	-
	2.00	5.84	0.629
	20.0	30.0	1.15

¹ Estimated since limit was established by saturator columns.

² Since time interval between sample collection events was consistent throughout study, the mean measured concentration represents an accurate estimation of exposure concentration.

³ Standard error of the mean.

* Limit of Detection (LOD) = 0.912 µ/L. Minimum Quantitation Level (LOQ) = 3.04 µ/L.

 

Mortality

No mortality was observed until hatch was completed on day 4. Little mortality was observed in the control and treatments during the 27-day post hatch period of the study. Larval and juvenile survival in the 5.84 and 30.0 μg/L treatments (mean measured concentrations) were not significantly different from the control (Mann-Whitney U test, p = 0.686 and 0.200 for 5.84 and 30.0 μg/L treatments, respectively). Survival rates in the control and the 5.84 and 30.0 μg/L treatments were 93.3, 91.7, and 90.0 %, respectively. Larval survival in the treatments was not significantly different from the control.

Hatching success

Hatching was observed from day 2 - 4 (end of hatch) in all 3 groups. The hatching success in the control, 5.84 and 30.0 μg/L treatments (mean measured concentrations) was 100 %. Therefore, no statistical analysis of this endpoint was performed.

Table 2. Effect of test item exposure on 30-Day Survival and Hatching Success¹

		Overall Survival (%)					Hatching Success (%)
Target Concentration (µg/L)	Replicate	N	Percent	Mean	SEM	CV(%)	N	Percent	Mean	SEM	CV(%)
0.0	A B C D	30 30 30 30	93.3 93.3 90.0 96.7	93.3	1.4	2.9	30 30 30 30	100.0 100.0 100.0 100.0	100.0	0.0	0.0
2.00	A B C D	30 30 30 30	86.7 93.3 93.3 93.3	91.7	1.7	3.6	30 30 30 30	100.0 100.0 100.0 100.0	100.0	0.0	0.0
20.0	A B C D	30 30 30 30	90.0 86.7 93.3 90.0	90.0	1.4	3.0	30 30 30 30	100.0 100.0 100.0 100.0	100.0	0.0	0.0

¹Survival on SD 30 of fertile embryos inserted on SD 0. SEM = Standard error of the mean. CV(%) = Coefficient of variation = (standard deviation / mean) x 100. Mean, SEM and CV(%) calculated from the percent survival estimate for each replicate within treatment (i.e. N = 4)
CV represents inter-replicate CV at each sampling point based on mean and standard deviation of each replicate (A - D) for each test concentration.

Appearance and behaviour

No significant effects on behavior or signs of overt toxicity were noted. In all control and treatment replicates on each exposure day normal appearance and behaviour was observed.

Validity criteria fulfilled:

yes

Conclusions:

In this test, survival above 90 % in all replicates and 100 % hatching success was observed throughout the exposure period. Therefore, the NOEC was determined to be >=30 µg/L based on mean measured concentration.

Executive summary:

A range-finder test for FSDT study (OECD 243) was performed following the conditions of OECD 210 with modifications. The test was conducted over 30 days post-fertilisation of the Danio rerio eggs. Mortality, appearance, behaviour and hatch success was observed throughout the exposure period. This test is considered acceptable to cover the long-term toxicity to fish endpoint.

Four replicates of 30 embryos each were used to initiate the test following the methods cited in OECD 210 with modification. A flow-through diluter system was used in the performance of the FSDT range finder. Exposure tanks were 10 L glass aquaria. The flow rate to each tank was 2.7 L/h (6.5 volume exchanges/d). Fluorescent lighting was used to provide a photoperiod of 16 h light and 8 h dark. Water temperature was maintained at 26 ± 1.5 °C, pH maintained between 7.3 to 7.9, and the dissolved oxygen (DO) concentration ≥ 4.9 mg/L (> 60 % of the air saturation) in each test tank. Thirty embryos per replicate tank, and 4 replicates per treatment (120 embryos/treatment) during exposure phases were used. Embryos were from a single clutch, double-selected using a dissecting microscope to ensure fertilization and normal appearing development. Exposure was started within 4 h after fertilization. Two test concentrations: 1) limit of solubility of the test substance under test conditions as produced by saturator columns (assigned a value of 20.0 μg/L nominal), and 2) a 10-fold dilution of the limit of solubility (assigned a value of 2.0 μg/L nominal) were evaluated. The exposure phase was conducted for 30 days post-fertilization (dpf). All exposure condition parameters were evaluated at pre-exposure phase d 0, in-life exposure phase d 0, and in-life test termination. Samples for analytical monitoring were taken on study days 0, 7, 14, 21, and test termination and were analysed after centrifugation.

The mean result of analyses of centrifuged samples from the recirculating saturator columns over a nine-day period were 14.7 ± 0.912 μg/L test item. Based on these results, the target concentration was set at 20.0 μg/L.

The mean measured concentrations (SEM) for the 0.0 (control), 2.00, and 20.0 μg/L treatments (initial target concentrations) were <LOD, 5.84 ± 0.629, and 30.0 ± 1.15 μg/L.

Survival rates in the control, 5.84 and 30.0 μg/L treatments were both 93.3, 91.7, and 90.0 %, respectively. Larval survival in the treatments was not significantly different from the control.

No significant effects on behaviour or signs of overt toxicity were noted. In addition, hatching success was 100 % in the control and each treatment.

In conclusion, since no significantly adverse effects were observed at the limit of water solubility, it will serve successfully as the maximum tolerated concentration (MTC) for the subsequent definitive FSDT study. The NOEC based on survival and hatch success was determined to be >=30 µg/L, respectively.

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2019-07-05 to 2020-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 243 - Fish Sexual Development Test

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

LC-MS/MS

Details on sampling:

Test solution from a representative replicate tank at the control and the test concentration was sampled for chemical analysis during the equilibration phase at least once. Test solution from each replicate tank in the limit concentration and control was sampled for chemical analysis on study day (SD) 0 and on in-life study conclusion (end of the eposure period). Thus, during the remainder of the in-life study samples were collected from one replicate tank of the control and limit concentration. The replicate selected for weekly sampling rotated (week 1 – A, week 2 – B, week 3 – C, week 4 –D, and repeat) throughout the in-life phase until the in-life phase was completed. Test solutions (20 mL) were collected in duplicate, centrifuged (2795g or 5000 rpm for 30 min) and subsequently diluted with 20 mL acetonitrile (ACN). Samples were stored refrigerated (ca. 4 °C) in 40 mL glass vials. The duplicate sample was retained and stored at 4 °C (1 - 9 °C) to be used as necessary in the event of the need for re-analysis.

Vehicle:

no

Details on test solutions:

Since the range finding study did not indicate substance-related effects at the water solubility limit under test conditions, a limit test design was selected. Therefore, the test concentration series was a limit test consisting of 1x and 0.00x (control), where x was the water solubility limit value. Based on a target MTC of 50.0 μg/L (centrifuged samples), the target test concentrations were 50.0 and 0.00 (control) μg/L.

- Preparation of saturator columns:
Eight saturator columns were prepared, two for control groups and six for preparation of test item stock solutions. In general, columns were prepared by 125 g/arm pyrolytically cleaned glass wool into each arm of u-shaped glass columns (diameter = 1.54 cm, length = 152.4 cm). To further clean the glass wool, the glass wool was subsequently rinsed with acetone and the acetone purged from the system prior to test substance loading. For the columns used to prepare test substance stock solution, approximately 10 g of the test item was dissolved in 30 mL acetone and distributed equally to both arms of the column. A vacuum pump was used to pull the solution through the glass wool to provide an even coat. The acetone was then evacuated from the columns leaving the test item. Once the saturator columns were dried and the acetone evacuated, the columns were plumbed, and dechlorinated tap water pumped through them at a rate of 4 mL/min for 10 days to rinse the columns and remove any glass particles from the glass wool.

- Determination of the water solubility:
Prior to in-life testing, water solubility was determined at test conditions. On three consecutive days, three samples were collected, centrifuged (2795 x g or 5000 rpm for 30 min.), and preserved. After collection, samples were stored at 4 °C (1 - 9 °C). Once all samples were collected, centrifuged, and preserved, samples were analysed. Three primary factors were used to determine if saturation was achieved: 1) in-life test conditions were maintained throughout assessment, 2) differences between three consecutive samples was ≤20 %, and 3) differences between centrifuged and uncentrifuged samples was ≤20 %. When each of the three criteria were met, the mean concentration of the three consecutive samples was considered saturation or the practical water soluble concentration, and the in-life study commenced.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: Embryos were from breeding of adults from an in-house culture. Adult fish were originally obtained from Aquatic Research Organisms (Hampton, NH, USA)
- Brood stock selection:
The exposure phase was initiated with newly fertilized embryos produced from adult fish from a laboratory supply of reproductively mature animals cultured at 26 ± 1°C. The fish were actively spawning. Test fish were selected from a single laboratory stock acclimated for at least two weeks prior to the test under conditions of water quality and illumination similar to those used in the test. Brood stock fish were obtained from an in-house culture, as shipping of adult fish is stressful and will interfere with reliable spawning. Apparently healthy looking parental fish were used for the breeding. Parental fish did not receive treatment for disease in the two-week period preceding breeding. Moribund fish or fish with clinical signs of disease were not used to produce the embryos used in the study. Since fertilized eggs needed to be obtained from at least 3 clutches, 6 pair of zebrafish were bred concurrently so that egg laying and fertilization occurred as synchronously as possible. One false bottom egg crate was placed in each tank to separate breeding fish from eggs. During the egg collection phases, eggs were cultured in a separate brood chamber.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
The addition of larvae to each treatment tank at test initiation used a random assignment process. Fertilization, stage, and normalcy of development were ascertained using a dissecting microscope. Since the random assignment process spanned a period of time, the stages at the beginning of assignment and end of assignment were monitored. Since exposure was required as early as possible after fertilization before cleavage of the blastodisc commenced the following procedure was used:
1. Determination of egg clutches that were of sufficient quantity and quality. Fertilization (≥90 %), development stage (ca. 4 hpf), and rate of normal development (≥90 %) was determined using a dissecting microscope. Approximately, 240 normal-appearing embryos from 3 clutches were used to initiate the study.
2. Once the clutches to be used were identified, they were pooled.
3. Pooled embryos were subsequently sorted to remove unfertilized or abnormal embryos. The number of pooled embryos exceeded 240.
4. Random assignment of embryos to tanks commenced and finished when 30 embryos were assigned to each tank (3 rounds of 10 embryos/round). The developmental stage at the time of first allocation and final allocation for each tank was noted. A representative sample (voucher) was collected at the beginning and conclusion of random assignment. The sub-sample of voucher embryos was preserved in 10 % neutral buffered formalin (NBF) to verify the stage of development at test initiation. The stage of development at the initiation of embryo assignment was 6 and 7 at the conclusion of assignment to the exposure tanks.

POST-HATCH FEEDING
- Type/source of feed: brine shrimp nauplii
- Amount given: ad libidum
- Frequency of feeding: twice per day
- Uneaten food and fecal material were removed from the test vessels daily 30 minutes after the last feeding by carefully cleaning the bottom of each tank using suction. Feeding was stopped 24 h before end of the test.

Test type:

flow-through

Water media type:

freshwater

Remarks:

Dechlorinated (DeCl2) tap water

Limit test:

yes

Total exposure duration:

63 d

Remarks on exposure duration:

ca. 63 days (until day 60 after end of hatch)

Hardness:

84 - 120 mg/L as CaCO3

Test temperature:

25.8 - 27.1 °C

pH:

6.7 - 7.6

Dissolved oxygen:

5.9 - 8.1 mg/L

Conductivity:

376 - 567 µS/cm

Nominal and measured concentrations:

Nominal concantrations: 0 (control) and 50 µg/L
Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: glass tanks in a flow-through system
- Material, size, headspace, fill volume: The system had water-contact components of glass (aquaria), stainless steel (diluter housing and water bath), and Teflon (tubing responsible for test material delivery). Exposure tanks were glass aquaria (with approximate measurements of 22.5 x 14.0 x 16.5 cm deep) equipped with standpipes that result in an actual tank volume of 10.0 L and minimum water depth of 10 to 15 cm.
- Aeration: none
- Type of flow-through: flow-through diluter system - Benoit Mini-Diluter
- Renewal rate of test solution (frequency/flow rate): 2.7 L/min, ca. 6.5 tank volumes/day
- No. of organisms per vessel: 30 freshly fertilised eggs (embryos)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Vehicle control performed: no
- Biomass loading rate: At 28 dpf (SD 28), the number of fish per replicate was assessed to determine if each replicate contains as equal a number of fish as possible. The loading rate in the control ranged from 0.84 g/L to 1.19 g/L and 0.32 g/L to 0.55 g/L in the test item treatment at the end of exposure. Since the difference between the number of fish in each replicate of the control or the test item treatment at 28 dpf was ≤ 4 test organisms, no re-distribution was performed.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated (charcoal-filtered) tap water prepared at the laboratory usinf 4-filter system - a multimedia filter to remove suspended solids in the feed water; a 10” pre-treatment filter (5 µm) to remove any additional solids; a 0.1 m³ activated virgin carbon treatment filter to remove chlorine, ammonia, and higher molecular weight organics; and a 5 µm polishing filter to remove any carbon particles from the carbon treatment phase.
- Total organic carbon: <0.3 - 1.3 mg/L
- Alkalinity: 36 - 64 mg/L as CaCO3
- Iodide: 8.1 - 8.3 µg/L
- Ammonia: <0.06 µg/L
- Chlorine: <0.05 µg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: twice a month

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 570 - 946 lux

EFFECT PARAMETERS MEASURED:
- survival,
- behavior of hatchlings and juveniles,
- body weights and length,
- VTG concentration,
- sex ratios,
- histopathology.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Limit test is generally accepted. No effects were observed in the reange-finding study, therefore, a limit test at the water solubility of the test substance under the test conditions was perfomed.

RANGE FINDING STUDY
- Test concentrations: 0 (control), 2.0 and 20.0 µg/L
- Results used to determine the conditions for the definitive study: In the range-finding study, survival above 90 % in all replicates and 100 % hatching success was observed throughout the exposure period. thus, a limit test at the water solubility limit was chosen for the definitive test.

Reference substance (positive control):

no

Key result

Duration:

63 d

Dose descriptor:

LOEC

Effect conc.:

46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

< 46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

63 d

Dose descriptor:

LOEC

Effect conc.:

46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

< 46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: sex ratio

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

< 46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

gonadal histology

Remarks:

and hepatotoxicity

Duration:

63 d

Dose descriptor:

LOEC

Effect conc.:

46.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

gonadal histology

Remarks:

and hepatotoxicity

Determination of practicle water solubility

Three consecutive samples met the three primary factors used to determine if saturation had been achieved: 1) in-life test conditions maintained throughout assessment, 2) difference in concentration ≤20 %, and 3) differences between centrifuged and uncentrifuged ≤20 %. The difference between each of the three consecutive samples relative to the mean ranged from 1.9 - 5.7 %. The difference between centrifuged and uncentrifuged samples for the three consecutive samples ranged from 7.3 - 11.6 %. The mean concentration based on the three consecutive centrifuged samples was 48.6 μg/L test item. Based on these results, the target concentration was set at 50.0 μg/L test item.

 

Water quality measurements and test system performance

Iodide (I-) levels in the dilution water were measured on SD 0 and 63 (in-life conclusion) of the study and contained 8.1 ± 0.08 and 8.3 ± 0.03 μg/L I-, respectively. All physicochemical water quality parameters in study were within acceptable ranges.

 

Confirmation of test concentrations

Target OMC concentrations selected for the FSDT definitive study were 0.0 (control) and 50.0 μg/L. The dilution water control solutions showed no detectable levels of the test item (<3.04 μg/L limit of quantitation, LOQ). Dilution water control samples collected during the study contained < LOD (LOD = 0.912 μg/L). Since time interval between sample collection events was consistent throughout study, the mean measured concentration represented a reasonable estimation of exposure concentration. The mean measured concentration represented the average of each data point from SD 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 for each replicate on SD 0 and 63; and sequential sampling of each replicate (A-D) of the control and each treatment per facility SOP. Interquartile ranges (IQRs) determined for the 0.0 (control), and 50.0 μg/L treatment were 0.46 - 0.46 and 24.15 - 61.35 μg/L test item, respectively. Based on IQR analysis of the control and each treatment group, five outliers were identified, original sample 50.0 μg/L replicates A-D SD 0 (8.26, 7.91, 7.30, and 7.75 μg/L test item), and replicate B SD 7 (22.8 μg/L test item). In this case, results of the duplicate samples of 50.0 μg/L replicates A-D (40.5, 40.3, 44.4, and 44.8 μg/L test item) on SD 0 and replicate B (47.4 μg/L test item) on SD 7 were used in the analysis. The corresponding mean measured concentrations in the definitive study were <LOD and 46.9 μg/L test item, respectively. The coefficient of variation (CV) [(Standard deviation/mean)100] was based on the standard deviation of the four replicate means (n = 4) for the control and the four replicate means (n = 4) for each treatment per facility SOP. The CVs of the intra-replicate means of the measured test concentrations for the control and 50.0 μg/L test item treatment were 0.0 % and 5.2 %, respectively. Each sample was within 20 % (35.5 - 56.3 μg/L test item) of the mean measured OMC concentration which were acceptable based on the criteria established in the test guidance and the study plan.

 

Mortality

No mortality was observed until hatching was completed on SD 4. Mean survival in the control and 46.9 μg/L treatment (mean measured concentration) were 82.5 % and 81.7 %, respectively. Survival in the 46.9 μg/L treatment was not significantly different from the control (t-test, p = 0.654).

 

Hatching success

Hatching was observed from SD 2-4 (end of hatch) in both the control and the test item treatment. The hatching success in the control and the treatment was 100 %. Therefore, no statistical analysis of this endpoint was performed.

 

Sex ratio

Sex ratios of control fish was 51.5 : 44.4 : 4.0 (%Female [F] : %Male [M] : %Undetermined [U]). Phenotypic sex ratios of fish exposed to 46.9 μg/L test item (mean measured concentration) was 55.1 : 34.7 : 10.2 (%F : M : U). Phenotypic sex ratio in the treatment was not significantly different from the control (t-test, p = 0.683 female and p = 0.174 male). In addition, the incidence of undetermined phenotypic sex (undifferentiated gonad) was not significantly different from the control (Mann-Whitney Rank Sum test, p = 0.057) indicating that the occurrence of primary sexual differentiation was not altered by the test item exposure in the present study.

 

Growth

Standard Length

Mean standard lengths were 22.1 mm in control female zebrafish and 15.1 mm in female zebrafish exposed to 46.9 μg/L test item (mean measured concentration). Mean standard lengths were 19.4 mm in control male zebrafish and 15.7 mm in male zebrafish exposed to the test item. Mean standard lengths were 15.1 mm in control undifferentiated zebrafish and 14.4 mm in undifferentiated zebrafish exposed to 46.9 μg/L test item. Standard lengths in both female and male fish exposed to the test item were significantly less than the control (t-test, p < 0.001 and p < 0.006, respectively). Standard length in undifferentiated fish exposed to the test item was not significantly different from the control (Mann-Whitney Rank Sum test, p = 0.800).

 

Body Weight

Mean body weights were 194.6 mg in control female zebrafish and 79.9 mg in female zebrafish exposed to the test item. Mean body weights were 132.0 mg in control male zebrafish and 83.3 mg in male zebrafish from the treatment group. Mean body weights were 169.8 mg in control undifferentiated zebrafish and 70.3 mg in undifferentiated treated zebrafish. Body weights in female and male fish exposed to the test item were significantly less than the control (Mann-Whitney Rank Sum test, p = 0.029 and t-test, p = 0.002). Body weight in undifferentiated fish exposed to the test item was not significantly different than the control (Mann-Whitney Rank Sum test, p = 0.133). Overall, the effects on growth occurred after the onset of gonadal differentiation.

 

Plasma Vitellogenin (VTG)

Mean plasma VTG levels were 571485.3 ng/mL in control female zebrafish and 564631.1 ng/mL in female zebrafish exposed to 46.9 μg/L test item (mean measured concentration). Mean plasma VTG levels were 255.4 ng/mL in control male zebrafish and 252.3 ng/mL in male zebrafish exposed to the test item. Plasma VTG LOQs were 4800.0 and 4.8 ng/mL for female and male zebrafish, respectively. Plasma VTG levels in female and male fish exposed to 46.9 μg/L test item were not significantly different from the control (t-test, p = 0.880 and Mann-Whitney Rank Sum test, p = 0.114, respectively). Plasma VTG levels in undifferentiated fish exposed to the test item were not significantly different from the control (Mann-Whitney Rank Sum test, p = 0.533).

 

Histophatology

Kidney

There were no exposure-related effects in the kidney.

 

Liver

Exposure-related effects in the liver included hepatocyte karyomegaly (minimal to moderate), oval cell proliferation (mild to moderate), single cell necrosis (minimal to mild), and increased mitotic figures (minimal). These findings were not distinct in undifferentiated or hermaphroditic fish.

Hepatocyte karyomegaly was characterised by nuclear enlargement with only modest cytoplasmic enlargement. Karyomegalic nuclei, which were oval or irregular in appearance, were frequently observed in paired or binucleate cells, and were typically hyperchromatic with prominent nucleoli. Because slight anisokaryosis noted as a variation in nuclear size was evident in the livers of occasional control fish, diagnoses of karyomegaly were not recorded unless affected nuclei were at least twice the diameter of average hepatocyte nuclei in the same liver section. The prevalence and severity of karyomegaly in the 46.9 μg/L test item-exposed males and females were comparable, and this finding was not observed in control fish. Oval cell proliferation occurred in three test item-exposed females. Oval cell proliferation was characterised by the presence of small cells with ovoid hyperchromatic nuclei and scant amounts of faintly basophilic fibrillar cytoplasm, which coursed through the liver in short chains or small clusters. Single cell necrosis was observed occasionally in the livers of control fish of both sexes, but the prevalence of this finding increased substantially in the test item-exposed males and females relative to control. Necrotic cells were small, round or irregular, with eosinophilic cytoplasm and pyknotic or karyorrhectic nuclei, consistent with an apoptotic form of cell death. Necrotic cells were typically surrounded by a clear halo and most often occurred within the cytoplasm of sinusoidal macrophages or other hepatocytes (phagocytosis). Increased mitotic figures occurred in only a single test item-exposed female; however, as that particular female additionally had mild hepatic karyomegaly, increased mitotic division was interpreted as further evidence of a regenerative response to hepatic injury.

 

Gonads

Exposure-related effects in the gonads included decreased mean ovarian stage scores in test item-exposed fish relative to controls. While the majority of control females (32 of 52 specimens) had ovarian stage scores of 1.0, characterised as immature ovaries characterised by the presence of both cortical alveolar and perinucleolar phase oocytes; the majority of females exposed to the test item (36 of 54 specimens) had Stage 0.0 ovaries, in which the most developed oocytes were perinucleolar phase. Undifferentiated gonads occurred in 3 of the 99 control fish and 8 of 98 fish exposed to 46.9 μg/L test item, respectively. Two of the 98 fish exposed to the test item were hermaphroditic. Seven instances of “intersex” specimens were identified in the present study, four of which occurred in control fish, and three in test item-exposed fish. Specific findings consisted of testicular oocytes (minimal) in three control males; ovarian spermatogenesis (minimal) in one control and one test item-exposed female; and hermaphroditism (not graded for severity) in two exposed fish. The finding of testicular oocytes was recorded for gonads in which one or more perinucleolar oocyte was embedded within the germinal epithelium of tissue that was otherwise clearly testicular. Conversely, in gonads with ovarian spermatogenesis, the majority of examined gonads was specifically ovarian, with the exception of a small portion of spermatogenic tissue in a single section. In fish diagnosed as hermaphroditic, different segments of the same gonad appeared definitively testicular or ovarian. Hermaphroditic gonads were not staged, and because these fish could not be definitively sexed, they were tabulated as a separate category resulting in either male, female, hermaphrodite, and undifferentiated designations. An additional intersex-like finding recorded in two control males was the presence of a gonadal duct with a female phenotype. This developmental anomaly was characterised by attachment of the testis to the dorsal coelomic mesothelium at two sites creating an intervening space. It should be noted that immature testes in male fish typically have a single attachment site with no space.All of these intersex changes were specifically and clearly interpreted as different manifestations of juvenile hermaphroditism, which is a well-documented phenomenon in normal laboratory zebrafish. Consequently, none of these findings were considered to be related to the test item treatment.

 

Summary of Histopathological Findings

The liver findings of hepatocyte karyomegaly, oval cell proliferation, single cell necrosis (increased prevalence compared to controls), and increased mitotic figures in test item-exposed fish were interpreted as indications of hepatic toxicity. Oval cells are progenitors of hepatocytes and bile duct epithelial cells, and oval cell proliferation in particular is typically associated with substantial hepatocyte loss. However, delayed ovarian development observed in test item-exposed females, and increased prevalence of undifferentiated gonads, was most likely related to the treatment-induced decrease in somatic growth. Although test item exposure was associated with reduced growth (mean body weight and standard length) relative to controls in both sexes, female fish appeared to be affected to a greater degree than males. As might be anticipated, fish with undifferentiated gonads were generally among the smallest fish in the study.

The seven instances of “intersex” gonads identified in present study, of which four were identified in control fish, were not considered to be treatment-related. During early development (2-3 wpf), the majority of zebrafish have gonads that exhibit the female (ovarian) phenotype, which begin gradually transitioning to the male (testicular) phenotype at approximately 5 weeks post-fertilization (wpf). Thus, at ca. 8 wpf at which point the exposure concluded and fish were sacrificed in the present study, it is not surprising that remnants of this transitional phase were evident as male-dominated (testicular oocyte), female-dominated (ovarian spermatogenesis), or neither-dominant (hermaphroditism) forms of intersex. Most importantly, no treatment-related differences in sex ratio or the frequency of undifferentiated gonads were noted. The subtle, but statistically insignificant, increase in the ratio of females to males in the test item-exposed fish compared to controls was most likely a function of delayed transition from the male to female phenotype as opposed to treatment-induced gonadal feminization.

 

Clinical signs of toxicity

Clinical signs of toxicity were not observed during the conduct of the present study.

Validity criteria fulfilled:

yes

Conclusions:

Based on the observed results, the NOEC was <46.9 μg/L and the LOEC was 46.9 μg/L.

Executive summary:

Freshly fertilized zebrafish eggs were exposed to the limit of solubility of the test substance under test conditions and dilution water control in accordance with OECD 234. Prior to in-life testing, practical water solubility of the test item was determined at test conditions.

To prepare stock solution required for dilution to the test solution, eight saturator columns were prepared, two for control groups and six for preparation of test item stock solutions.

A flow-through diluter system (Benoit Mini-Diluter) was used in the performance of the FSDT exposure. The Benoit mini-diluter system had water-contact components of glass (aquaria), stainless steel (diluter housing and water bath), and Teflon® (tubing responsible for test material delivery). Exposure tanks were glass aquaria equipped with standpipes with an actual tank volume of 10 L. The flow rate to each tank was 2.7 L/h (6.5 volume exchanges/d). Fluorescent lighting was used to provide a photoperiod of 16 h light and 8 h dark at an intensity that ranged from 540 to 1000 lux (lumens/m2) at the water surface. Water temperature was maintained at 27 ± 2 °C and did not differ by more than ±1.5 °C between test chambers at any one time. In addition, pH was maintained between 6.5 to 9.0, and the dissolved oxygen (DO) concentration was maintained at ≥ 4.9 mg/L (> 60 % of the air saturation) in each test tank.

Temperature was measured daily; and pH, DO, and light intensity (lux) were measured two times per week in all control and treatment tanks. Total hardness, alkalinity, and conductivity were measured in all chambers at in-life initiation and conclusion. Iodine as I- was measured in the dilution water on life initiation and conclusion. TOC was measured in the dilution water weekly throughout the in-life phase.

Test solution from each replicate tank in the limit concentration and control was sampled for chemical analysis on SD 0 and on in-life study conclusion. Thus, during the remainder of the in-life study samples were collected from one replicate tank of the control and limit concentration. The replicate selected for weekly sampling rotated (week 1 – A, week 2 – B, week 3 – C, week 4 –D, and repeat) throughout the in-life phase until the in-life phase was completed.

Thirty embryos per replicate tank, and 4 replicates per treatment (120 embryos/treatment) during exposure phases were used. Embryos from three clutches were double-selected using a dissecting microscope to ensure fertilization and normal appearing development and mixed prior to addition to replicate tanks. Exposure was started within 4 h after fertilization. The test chemical was delivered to the exposure chambers using a continuous-flow diluter. The exposure phase was conducted for 60 days post-hatch (dph). All exposure condition parameters were evaluated at pre-exposure phase d 0, in-life exposure phase d 0, and in-life test termination. Samples for analytical monitoring were collected on SD 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 (in-life termination) and were analysed after centrifugation.

The following parameters were determined throughout the exposure period - survival (daily), body weights and length, behaviour of hatchlings and juveniles (daily), vitellogenin (VTG), sex ratios, histopathology of the kidney, liver and gonads.

Results of the practical water solubility analyses of centrifuged samples indicated that the mean concentration based on the three consecutive centrifuged samples was 48.6 μg/L test item. Therefore, the target test item concentrations selected for the FSDT study were 0.0 (control) and 50 μg/L. The corresponding mean measured concentration in the definitive study was 46.9 μg/L for the treatment and <LOD for the control. Mean survival in the control and the treatment was 82.5 % and 81.7 %, respectively. Mortality in the treatment was not significantly different from the control. The hatching success in the control and the treatment was 100 % and was complete by 4 days post-fertilization (dpf). Phenotypic sex ratio in the treatment was not significantly different from the control for both males and females. In addition, the incidence of undetermined (undifferentiated) phenotypic sex (undifferentiated gonad) was not significantly different from the control. Standard lengths in both female and male fish exposed to 46.9 μg/L test item were significantly less than those of the control group. Standard length in undifferentiated fish exposed to the test item was not significantly different from the control. Body weights in female and male fish exposed to the test item were significantly less than those of the control. Body weight in undifferentiated test item-exposed fish was not significantly different from the control. Plasma VTG levels in female and male fish exposed to the test item concentration were not significantly different from the control. Plasma VTG levels in undifferentiated fish exposed the test item were not significantly different from the control.

Exposure-related effects observed in the liver included hepatocyte karyomegaly (minimal to moderate), oval cell proliferation (mild to moderate), single cell necrosis (minimal to mild), and increased mitotic figures (minimal). These findings were not distinct in undifferentiated or hermaphroditic fish. The liver findings of hepatocyte karyomegaly, oval cell proliferation, single cell necrosis (increased prevalence compared to controls) and increased mitotic figures in 46.9 μg/L test item-exposed fish were interpreted as indications of hepatic toxicity. Exposure-related effects in the gonads included decreased mean ovarian stage scores in 46.9 μg/L test item-exposed fish relative to controls. However, the delayed ovarian differentiation observed in the treatment-exposed females was related to the treatment-induced decrease in somatic growth. The seven instances of “intersex” gonads identified (four of which were identified in control fish) were not considered to be treatment-related. The subtle, but statistically insignificant, increase in the ratio of females to males in the test item-exposed fish compared to controls was most likely a function of delayed transition from the male to female phenotype as opposed to treatment-induced gonadal feminization. No significant effects on behaviour or signs of overt toxicity were noted.

In conclusion, the present study indicated that exposure to 46.9 μg/L test item (< 20% CV from the practical solubility under test conditions) did not reduce survival, alter sex ratio, increase the proportion of undifferentiated gonads, or alter plasma vitellogenin levels relative to control zebrafish. However, both standard length and body weight were reduced in female and male fish, but not in undifferentiated fish exposed to 46.9 μg/L mean measured test item concentration. Early life stage exposure of zebrafish to 50 μg/L (target concentration) test item was associated with morphologic changes in the liver consistent with hepatotoxicity. It is reasonable to surmise that hepatotoxicity was responsible for the decreased somatic growth experienced by test item-treated fish, which in turn was likely the cause of delayed ovarian differentiation in females as determined by gonadal stage scoring. The low incidence of intersex in both control and test item-exposed fish, which manifested as either testicular oocytes, ovarian spermatogenesis, or hermaphroditism, was not considered to be related to the test item treatment, but was instead a function of gonadal immaturity in a species that is known to exhibit juvenile hermaphroditism. Based on these results, the NOEC was <46.9 μg/L and the LOEC was 46.9 μg/L. The highest tested concentration represented the limit of solubility under test conditions. However, it should be noted that the response observed was not related with an endocrine-mediated mode of action.

Description of key information

A Fish Sexual Development Test (FSDT) according to OECD 234 has been requested by ECHA, thus, the endpoint long-term toxicity to fish was covered by the results obtained during test. Additionally, the results from the non-GLP range-finder for the FSDT, which was performed according to OECD 210, were included. Based on survival rates and hatching success of Danio rerio eggs in the pre-test, the NOEC was determined to be >=30 µg/L. Based on the results and observations in the FSDT study, the NOEC was determined to be <46.9 µg/L and the LOEC to be 46.9 µg/L mean measured test item concentration.

Additional information

Key Study - OECD 243

Freshly fertilized zebrafish eggs were exposed to the limit of solubility of the test substance under test conditions and dilution water control in accordance with OECD 234. Prior to in-life testing, practical water solubility of the test item was determined at test conditions.

To prepare stock solution required for dilution to the test solution, eight saturator columns were prepared, two for control groups and six for preparation of test item stock solutions.

A flow-through diluter system (Benoit Mini-Diluter) was used in the performance of the FSDT exposure. The Benoit mini-diluter system had water-contact components of glass (aquaria), stainless steel (diluter housing and water bath), and Teflon® (tubing responsible for test material delivery). Exposure tanks were glass aquaria equipped with standpipes with an actual tank volume of 10 L. The flow rate to each tank was 2.7 L/h (6.5 volume exchanges/d). Fluorescent lighting was used to provide a photoperiod of 16 h light and 8 h dark at an intensity that ranged from 540 to 1000 lux (lumens/m2) at the water surface. Water temperature was maintained at 27 ± 2 °C and did not differ by more than ±1.5 °C between test chambers at any one time. In addition, pH was maintained between 6.5 to 9.0, and the dissolved oxygen (DO) concentration was maintained at ≥ 4.9 mg/L (> 60 % of the air saturation) in each test tank.

Temperature was measured daily; and pH, DO, and light intensity (lux) were measured two times per week in all control and treatment tanks. Total hardness, alkalinity, and conductivity were measured in all chambers at in-life initiation and conclusion. Iodine as I- was measured in the dilution water on life initiation and conclusion. TOC was measured in the dilution water weekly throughout the in-life phase.

Test solution from each replicate tank in the limit concentration and control was sampled for chemical analysis on SD 0 and on in-life study conclusion. Thus, during the remainder of the in-life study samples were collected from one replicate tank of the control and limit concentration. The replicate selected for weekly sampling rotated (week 1 – A, week 2 – B, week 3 – C, week 4 –D, and repeat) throughout the in-life phase until the in-life phase was completed.

Thirty embryos per replicate tank, and 4 replicates per treatment (120 embryos/treatment) during exposure phases were used. Embryos from three clutches were double-selected using a dissecting microscope to ensure fertilization and normal appearing development and mixed prior to addition to replicate tanks. Exposure was started within 4 h after fertilization. The test chemical was delivered to the exposure chambers using a continuous-flow diluter. The exposure phase was conducted for 60 days post-hatch (dph). All exposure condition parameters were evaluated at pre-exposure phase d 0, in-life exposure phase d 0, and in-life test termination. Samples for analytical monitoring were collected on SD 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 (in-life termination) and were analysed after centrifugation.

The following parameters were determined throughout the exposure period - survival (daily), body weights and length, behaviour of hatchlings and juveniles (daily), vitellogenin (VTG), sex ratios, histopathology of the kidney, liver and gonads.

Results of the practical water solubility analyses of centrifuged samples indicated that the mean concentration based on the three consecutive centrifuged samples was 48.6 μg/L test item. Therefore, the target test item concentrations selected for the FSDT study were 0.0 (control) and 50 μg/L. The corresponding mean measured concentration in the definitive study was 46.9 μg/L for the treatment and <LOD for the control. Mean survival in the control and the treatment was 82.5 % and 81.7 %, respectively. Mortality in the treatment was not significantly different from the control. The hatching success in the control and the treatment was 100 % and was complete by 4 days post-fertilization (dpf). Phenotypic sex ratio in the treatment was not significantly different from the control for both males and females. In addition, the incidence of undetermined (undifferentiated) phenotypic sex (undifferentiated gonad) was not significantly different from the control. Standard lengths in both female and male fish exposed to 46.9 μg/L test item were significantly less than those of the control group. Standard length in undifferentiated fish exposed to the test item was not significantly different from the control. Body weights in female and male fish exposed to the test item were significantly less than those of the control. Body weight in undifferentiated test item-exposed fish was not significantly different from the control. Plasma VTG levels in female and male fish exposed to the test item concentration were not significantly different from the control. Plasma VTG levels in undifferentiated fish exposed the test item were not significantly different from the control.

Exposure-related effects in the liver included hepatocyte karyomegaly (minimal to moderate), oval cell proliferation (mild to moderate), single cell necrosis (minimal to mild), and increased mitotic figures (minimal). These findings were not distinct in undifferentiated or hermaphroditic fish. The liver findings of hepatocyte karyomegaly, oval cell proliferation, single cell necrosis (increased prevalence compared to controls) and increased mitotic figures in 46.9 μg/L test item-exposed fish were interpreted as indications of hepatic toxicity. Exposure-related effects in the gonads included decreased mean ovarian stage scores in 46.9 μg/L test item-exposed fish relative to controls. However, the delayed ovarian differentiation observed in the treatment-exposed females was related to the treatment-induced decrease in somatic growth. The seven instances of “intersex” gonads identified (four of which were identified in control fish) were not considered to be treatment-related. The subtle, but statistically insignificant, increase in the ratio of females to males in the test item-exposed fish compared to controls was most likely a function of delayed transition from the male to female phenotype as opposed to treatment-induced gonadal feminization. No significant effects on behaviour or signs of overt toxicity were noted.

In conclusion, the present study indicated that exposure to 46.9 μg/L test item (< 20% CV from the practical solubility under test conditions) did not reduce survival, alter sex ratio, increase the proportion of undifferentiated gonads, or alter plasma vitellogenin levels relative to control zebrafish. However, both standard length and body weight were reduced in female and male fish, but not in undifferentiated fish exposed to 46.9 μg/L mean measured test item concentration. Early life stage exposure of zebrafish to 50 μg/L (target concentration) test item was associated with morphologic changes in the liver consistent with hepatotoxicity. It is reasonable to surmise that hepatotoxicity was responsible for the decreased somatic growth experienced by test item-treated fish, which in turn was likely the cause of delayed ovarian differentiation in females as determined by gonadal stage scoring. The low incidence of intersex in both control and test item-exposed fish, which manifested as either testicular oocytes, ovarian spermatogenesis, or hermaphroditism, was not considered to be related to the test item treatment, but was instead a function of gonadal immaturity in a species that is known to exhibit juvenile hermaphroditism. Based on these results, the NOEC was <46.9 μg/L and the LOEC was 46.9 μg/L. The highest tested concentration represented the limit of solubility in the test medium under test conditions. However, it should be noted that the response observed was not related with an endocrine-mediated mode of action.

Supporting Study - OECD 210

A range-finder test for FSDT study (OECD 243) was performed following the conditions of OECD 210 with modifications. The test was conducted over 30 days post-fertilisation of the Danio rerio eggs. Mortality, appearance, behaviour and hatch success was observed throughout the exposure period. This test is considered acceptable to cover the long-term toxicity to fish endpoint.

Four replicates of 30 embryos each were used to initiate the test following the methods cited in OECD 210 with modification. A flow-through diluter system was used in the performance of the FSDT range finder. Exposure tanks were 10 L glass aquaria. The flow rate to each tank was 2.7 L/h (6.5 volume exchanges/d). Fluorescent lighting was used to provide a photoperiod of 16 h light and 8 h dark. Water temperature was maintained at 26 ± 1.5 °C, pH maintained between 7.3 to 7.9, and the dissolved oxygen (DO) concentration ≥ 4.9 mg/L (> 60 % of the air saturation) in each test tank. Thirty embryos per replicate tank, and 4 replicates per treatment (120 embryos/treatment) during exposure phases were used. Embryos were from a single clutch, double-selected using a dissecting microscope to ensure fertilization and normal appearing development. Exposure was started within 4 h after fertilization. Two test concentrations: 1) limit of solubility of the test substance under test conditions as produced by saturator columns (assigned a value of 20.0 μg/L nominal), and 2) a 10-fold dilution of the limit of solubility (assigned a value of 2.0 μg/L nominal) were evaluated. The exposure phase was conducted for 30 days post-fertilization (dpf). All exposure condition parameters were evaluated at pre-exposure phase d 0, in-life exposure phase d 0, and in-life test termination. Samples for analytical monitoring were taken on study days 0, 7, 14, 21, and test termination and were analysed after centrifugation.

The mean result of analyses of centrifuged samples from the recirculating saturator columns over a nine-day period were 14.7 ± 0.912 μg/L test item. Based on these results, the target concentration was set at 20.0 μg/L.

The mean measured concentrations (SEM) for the 0.0 (control), 2.00, and 20.0 μg/L treatments (initial target concentrations) were <LOD, 5.84 ± 0.629, and 30.0 ± 1.15 μg/L.

Survival rates in the control, 5.84 and 30.0 μg/L treatments were both 93.3, 91.7, and 90.0 %, respectively. Larval survival in the treatments was not significantly different from the control.

No significant effects on behaviour or signs of overt toxicity were noted. In addition, hatching success was 100 % in the control and each treatment.

In conclusion, since no significantly adverse effects were observed at the limit of water solubility, it will serve successfully as the maximum tolerated concentration (MTC) for the subsequent definitive FSDT study. The NOEC based on survival and hatch success was determined to be 30 µg/L, respectively.