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Long-term toxicity to fish

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Reference
Endpoint:
long-term toxicity to fish, other
Remarks:
Fish Sexual Development Test, Flow through conditions
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
OECD guideline study with GLP documentation.
Qualifier:
according to
Guideline:
other: OECD test guideline, Sect. 2: Effects on Biotic Systems, N. 234 “Fish, Sexual Development Test”
Version / remarks:
adopted July 28, 2011
Deviations:
no
Principles of method if other than guideline:
One amendment was prepared during the course of the study. It described a re-start of the study due to low survival rates. It was suspected that increased mortality was due to reduced feed supply. Thus, it was decided to change the feeding regime in order to increase the food supply for the larvae. Furthermore, the larvae were retained in the fry chambers until 14 dpf before the release in the main test vessels.
GLP compliance:
yes
Specific details on test material used for the study:
Storage conditions: Do not store above 40°C.
Analytical monitoring:
yes
Remarks:
LC-MS/MS
Details on sampling:
SAMPLING
Samples of holding and dilution water were taken out of the flow-through system by analytical laboratory using piston operated pipettes.
For sampling at first 2.0 mL methanol were pipetted into screw top vials. Subsequent 10.0 mL of the water phase were taken out of the test vessels and were pipetted into the screw top vials as well; the volume ratio water/methanol was therefore = 5+1 (v+v).
After vigorously mixing by hand the samples were transferred to the analytical laboratory where they were analyzed without loss of time. If it was not possible to analyse samples directly after sampling, these samples were stored immediately after sampling at ≤ -20°C in a freezer.
Vehicle:
yes
Remarks:
Acetone
Details on test solutions:
PREPARATION OF STOCK SOLUTIONS
For preparation of the stock solution, an appropriate amount of Preventol A 6 was weighted out and dissolved in acetone, resulting in a final concentration of 36 mg test item/mL acetone. This solution served as initial stock solution for preparation of the acetonic stock solution 1 (1 mL acetonic stock solution 2 in a total volume of 10 mL acetone) and as acetonic stock solution 2. One mL of each acetonic stock solution was applied to the inner surface of a clean glass bottle. After complete evaporation of the solvent, the bottles were filled with 10 L of dilution water and stirred over night for approx. 24 hours. The aqueous stock solutions were transferred to stainless steel tanks and filled up to a final volume of 40 L. These solutions served as application solutions in the flow through device. To achieve the final concentration in the test vessels, the application solutions were mixed with dilution water in adequate volumes via the dosing pumps.

TEST ITEM CONCENTRATIONS
According to the CoA (see chapter A.8.1), the content of the active ingredient diuron in the test item Preventol A 6 (trade name of the a.i. diuron) is 98.7 %. Mean measured concentrations given in the report are thus based on nominal concentrations of the test item Preventol A 6, as this is considered as minor impurity.
The concentrations of the active a.i. diuron in the test item Preventol A 6 were assessed by chemical analysis using LC/MS-MS. The LOQ was set to 0.3 μg a.i. /L. At test start and after 7 days, samples were taken from all test vessels in order to confirm correct dosing. During the time course of the study, samples were taken on a weekly basis, alternating between the vessels that were served by the same dosage pump. At test end, samples of all vessels were analyzed.
Mean concentrations per treatment of the a.i. during the course of the study were between 102.9 % and 118.7 % of the nominal concentration of the test item (Table 7). As few samples differed from the desired 80 – 120 % of nominal, it was decided to base the biological effects on mean measured concentrations (1.19 μg/L; 3.26 μg/L; 11.32 μg/L; 32.51 μg/L; 105.44 μg/L).

SAMPLE PREPARATION
For chemical analysis the taken water samples (water/methanol mixtures) of the nominal test concentration 10, 31.6 and 100 μg/L were diluted with solvent mixture 1 to adjust the analyte concentrations to the calibration range of the measurement system. For this purpose, 1 mL of the taken water samples (water/methanol mixture) were pipetted into a volumetric flask (10 or 20 mL) and filled up to the mark with solvent mixture 1; the procedure is given Table 13.
The control and the taken water samples of the nominal test concentration of 1 and 3.16 μg/L remained undiluted.
After manual shaking 1.2 mL of the dilutions and 20 μL of the IS spiking solution were pipetted into 2 mL screw top vials and 10 μL was measured by LC-MS/MS as described in chapter A.2.4.

BLOOD SAMPLING
The fish blood was sampled by cardiac puncture after anesthetization of the animals. To avoid coagulation of blood and degradation of protein, the samples were collected within phosphate-buffered saline (PBS) buffer containing heparin (1000 units/mL) and the protease inhibitor aprotinin (2 TIU/mL). As ingredients for the buffer, heparin as ammonium-salt (Sigma) and lyophilized aprotinin (Roth) were used. For blood sampling a syringe (1 mL) with a fixed needle was used. The syringe was prefilled with buffer (approximately 300 μL) to completely elute the small blood volumes from each fish. Blood samples ranging from 15-40 μL were taken. Plasma was separated from the blood via centrifugation (30 min, 5000 g, 4°C) and immediately stored at -80°C until further analysis.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
SPECIFICATION
Species: Zebrafish (Danio rerio, Teleostei, Cyprinidae)
Source: Test facility bred
Holding: Fertilized eggs for the test were obtained from individuals reared in the laboratory of the Fraunhofer Institute, Schmallenberg, Germany
Origin of the used strain of zebrafish: West Aquarium GmbH, 37431 Bad Lauterberg, Germany

HOLDING OF PARENTAL FISH AND BREEDING CONDITIONS:
Parental fish (maximum age: 2 years) were held in aquaria with a total volume of 150 L. Holding water was of the same quality as it was used in the test (purified drinking water, chapter 4.2). Holding temperature was 26.0 °C ± 2.0 °C, the photoperiod is 12 h light / 12 h dark, the light intensity was approx. 1000 lux, 5 cm above water surface in the middle of each vessel. Flow through rate was adjusted to reach a 1-fold exchange of water per day. Animals were fed daily ad libitum with TetraMin® Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina). Fish larvae were fed with breeding food (Tetra Werke, Melle, Germany). The broodstock was visually checked every day for mortality, illness, parasites or abnormal behavior. No prophylactic treatment of fish was taken place. Only healthy fish without diseases and abnormalities were used as parental fish for the production of fertilized eggs.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
63 d
Remarks on exposure duration:
The study lasted until 63 days post fertilization (dpf).
Hardness:
1.1 - 1.2 mmol/L
Test temperature:
25.8 °C to 27.0 °C
pH:
7.98 - 8.30
Dissolved oxygen:
oxygen saturation between 84 % and 100 %
Conductivity:
267.0 - 278.0 µS/cm
Nominal and measured concentrations:
100 μg/L; 31.6 μg/L; 10.0 μg/L; 3.16 μg/L; 1.00 μg/L (nominal)
Details on test conditions:
TEST VESSELS
The test vessels were glass aquaria with a total volume of 28 L and approx. 25 L of test solution. Each replicate group was kept in an individual test vessel. Each test chamber was labelled with the study number, the test item and test concentrations. At test start, each test vessel was equipped with two fry cages, being glass cylinders with a brim height of 10 cm and a diameter of 8 cm. The bottom of each cage was a Teflon gaze with a pore size of 0.4 mm.

TEST CONDITIONS
The light regime was 12 hours light / 12 hours dark (light intensity of approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel). Water temperature was maintained by placing the vessels into a temperature controlled water bath set to 27.0 °C ± 2.0 °C. The oxygen concentration of the test solution was not lower than 60 %.

TEST CONCENTRATIONS
The concentration range was discussed and agreed with the sponsor before test start. In detail, it is based on available data from fish studies with the same chemical on acute and chronic toxicity, i.e. acute LC50 and LOEC values. It has to be mentioned that available data was raised from different fish species, being rainbow trout and fathead minnow. For fathead minnow, an LC50 value of 14.2 mg diuron/L was determined within a 96-h acute fish study.
LC50 values for acute and long term toxicity of diuron to rainbow trout were determined to be 14.7 mg/L and 4.01 mg/L, respectively. In this context, is has to be mentioned, that a lethal threshold concentration was determined to be 1.47 mg/L. In the study on prolonged toxicity, the lowest concentration in which lethality was observed was 0.79 mg/L.
The guideline recommends that concentrations higher than 10 % of the acute adult LC50 need not to be tested. Furthermore, it is recommended that the maximum test concentration should be in the range of 10 % of the LC50 on larval life stage. It was decided to choose test concentrations slightly below this level to ensure the absence of systemic toxicity effects in the study.
Furthermore, within a study with fathead minnow on chronic toxicity of early life stages, a LOEC of 61.8 μg diuron/L for the endpoint survival was determined. As fathead minnow also belongs to the family of cyprinids, this LOEC is considered as most relevant to set the final concentration range in the FSDT.
Based on the available data, the highest test concentration was set to 100 μg diuron/L. A spacing factor of √10 was applied in order to cover a preferably wide concentration range.
The full concentration range of diuron, applied as the test item Preventol A 6 in the FSDT was set as follows:
100 μg/L; 31.6 μg/L; 10.0 μg/L; 3.16 μg/L; 1.00 μg/L.

FLOW THROUGH SYSTEM
For two replicate vessels each, an individual dosage system was used, i.e., two dosage systems for each concentration plot. Dilution water was pumped by a water dosage pump (membrane pump, Prominent, Heidelberg, Germany) into a mixing chamber, placed on a magnetic stirrer. An adequate amount of the stock solution was added into the magnetic stirrer via a stock solution dosage pump (membrane pump with a stainless steel head, Prominent, Heidelberg, Germany). The prepared test solution flowed into the test vessels via flexible tubes, distributed to the two vessels by an electronically regulated distributor driven. The dilution water control was served by dilution water only.
Controls and all test concentrations were run in four replicate aquaria, each. Thus, each treatment plot consisted of four replicates. For every test vessel a water flow rate of five volumes per vessel and day was adjusted. The maximum loading rate for zebrafish specified in the guideline was respected.

TEST PERFORMANCE
Oxygen concentration and pH were measured in each test vessel directly before adding the eggs and afterwards at least twice per week. The water temperature was measured each working day in all test vessels and in the control. Additionally, the water temperature was measured continuously in at least two control vessels. Uneaten food and faeces was removed from the fry cages and the test aquaria at regular intervals.
At test start 2 x 15 fertilized and randomized eggs (for egg collection see chapter 4.1.4) were placed in the glass beakers serving as fry cages. To allow better observation of eggs and larvae, the glass beakers were placed in the mid part of each test vessel (see chapter 4.3.1). Each aquarium was equipped with two fry cages. 120 eggs (4 x 30) were used for each test concentration. From day 7 on (dpf = days post fertilization), larvae were fed once daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany). From the same date on, brine shrimp nauplii (Artemia salina) were added twice daily and. From day 14 (dpf) on, ground TetraMin flakes were added once daily to the fish feed. The larvae were retained in the fry chambers until 14 dpf before the release in the main test vessel.
After 21 and 35 days, survival was determined by photographic counting. After 35 days, length of each individual fish was measured by digital photography. At test end (day 63), all fish were sacrificed and the individual lengths and weights were measured.
At termination of the fish groups, the fish were prepared as follows: the fish were anaesthetized using chloro-butanol. A blood sample was taken from each fish afterwards via heart puncture. After determination of length and weight, the fish were killed with a dorsal cut. All fish were transferred to an appropriate fixative to allow a histopathological analysis of fish tissue.

BLOOD COLLECTION AND PREPARATION
To avoid coagulation of blood and degradation of protein, the samples were collected within phosphate-buffered saline (PBS) buffer containing heparin (1000 units/mL) and the protease inhibitor aprotinin (2TIU/mL). As ingredients for the buffer, heparin as ammonium-salt (Sigma) and lyophilised aprotinin (Roth) was used. For blood sampling a syringe (1 mL) with a fixed needle was used. The syringe was prefilled with buffer (approximately 300 μL) to completely elute the small blood volumes from each fish. Blood samples ranging from 15-40 μL were taken by cardiac puncture. At first the fish were anaesthetised with chloro-butanol (5 g/L). Plasma was separated from the blood via centrifugation (30 min; 5000 g; 4°C) and immediately stored at -80 °C until further analysis.

FIXATION, EMBEDDING AND SECTIONING (NON-GLP)
The samples, which were chosen for histological investigations, were transferred into tissue cassettes which were labelled according to the internal coding system of the pathology laboratory. The cassettes were placed into the baskets of a tissue processor and were dehydrated. Afterwards, they were transferred to the embedding station, properly oriented and embedded in paraffin. For sectioning, the paraffin blocks were placed into the chuck of a rotating microtome in a way that transversal sections proceeding from the ventral side to the dorsal side of the fish were obtained. After trimming of the block, sections at 5 μm thickness were prepared. From each fish, a total of three step sections were prepared and mounted on glass slides. The first section was taken at the point where approx. a third to a half of the gonad is removed; the following two sections were taken at 50 - 100 μm distance. The slides were dried, placed into an automated slide stainer and stained using haematoxylin-eosin.
After evaluation of the gonads, the number code was used to associate the fish with the treatment levels and sampling intervals. The overall embedding and sectioning procedures followed the OECD Guidance document on histology and histopathology guidelines.
All examined individuals were staged with respect to the maturation status of their gonads. The staging was done according to the OECD Guidance document. The occurrence of intersex was recorded. If possible, the results were statistically evaluated.
Reference substance (positive control):
no
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
1 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks:
post-hatch survival
Remarks on result:
other: The study lasted until 63 days post fertilization (dpf).
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
1 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: The study lasted until 63 days post fertilization (dpf).
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: endocrine-related endpoint
Remarks on result:
other: The study lasted until 63 days post fertilization (dpf).
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: endocrine-related endpoints
Remarks on result:
other: The study lasted until 63 days post fertilization (dpf).
Details on results:
No effects were determined for the endpoints growth in terms of length and weight.
Furthermore, no effects were determined for the endocrine-related test endpoints sex ratio and VTG content in female and male blood plasma. Also histopathological evaluations showed no indicative effects on gonad maturation or lesions.
Reported statistics and error estimates:
All biological response data were analyzed and reported separately by sex, if appropriate. For each endpoint, the NOEC and LOEC were determined. All statistics were calculated using ToxRat® Professional 3.2.1.
No Observed Effect Concentrations (NOEC) were calculated, using ANOVA, followed by a non-parametric approach, based on rank sums (e.g. Jonckheere-Terpsta test suggested in the guideline), as this test does not require transformation of quantal data (e.g. post-hatch survival, sex ratio) and could be applied to each endpoint, resulting in better comparability of statistical results.
Details of all statistical analyses were reported, including exact p-values for all statistical comparisons.
Results of all tests for normality and homogeneity of variance were reported along with the results of the final test.

BIOLOGICAL ENDPOINTS

Early life stage

Hatching rate

First larvae hatched at 3 dpf across all treatment levels. Hatch was completed at 6 to 8 dpf in all replicates, with no difference between treatments. Single eggs were found coagulated during this period. 96.7 to 100.0 % hatch was observed in all replicates.

Post-hatch survival

Survival was recorded daily on a visual basis and at 21 and 35 dpf by photographic counting. Between 11 dpf and 21 dpf, increased larval death was observed across test levels. Mortality of larvae thus occurred mainly before 21 dpf, during the phase of transition from yolk sac feeding to external feeding. Surviving larvae did not display any signs of disease. At 21 dpf, the post-hatch survival rate in controls reached a mean value of 83.1 %. At 35 dpf, post-hatch survival in control vessels was at 82.3 % thus met the validity criterion for survival in controls of ≥ 70 %. Statistical analyses of post-hatch survival at 35 dpf revealed significant differences between control and treatments at concentrations ≥ 3.26 μg a.i./L (Jonckheere-Terpstra Test, p<0.05; one-sided smaller). Thus, the NOEC for the endpoint post-hatch survival during ELS was defined at 1.19 μg a.i./L (mean measured concentration).

Length at 35 dpf

At 35 dpf, larval growth in terms of length was determined. In this study, mean length of 1.60 cm was observed in controls. The mean length of larvae in the treatment conditions were found in the range of 1.57 cm (1.19 μg/L) and 1.80 cm (32.51 μg/L).

Statistical analyses revealed no significant decrease in size compared to control values (Jonckheere-Terpstra Test, α=0.05; one-sided smaller). Thus, the NOEC based on the endpoint size in terms of length after the ELS was defined as ≥ 105.44 μg a.i./L (mean measured concentration).

Test termination

Survival at 63 dpf

One juvenile fish each at mean measured concentrations of 11.32 and 32.51 μg a.i./L were found dead during the JG phase. No other signs of disease were observed. Statistical analyses of post-hatch survival at 63 dpf revealed significant differences between control and treatments at concentrations ≥ 3.26 μg a.i/L. (Jonckheere-Terpstra Test, p<0.05; one-sided smaller). Thus, the NOEC for the endpoint post-hatch survival at test end was defined at 1.19 μg a.i./L (mean measured concentration). However, the adverse effect on post-hatch survival at test end was a result of the decreased survival rate during the ELS phase. No test item-related effect on post-hatch survival between 35 dpf and test end was observed.

Length and weight at 63 dpf

At 63 dpf, growth in terms of length was determined. Juvenile fish in the control displayed a mean measured length of 2.9 cm. The mean length of fish under treatment conditions varied between 3.0 cm and 3.4 cm, with no concentration-response relationship. The mean measured weight of fish in controls was measured at 0.235 g, the mean weight of fish under treatment conditions varied between 0.261 g and 0.414 g, again with no concentrationresponse relationship. Fish exposed to the test item were larger and gained weight compared to controls. However, this effect is likely due to lower density due to the decreased number of surviving fish and is thus not directly substance-related. Furthermore, increased size is not considered as negative effect and rather promotes fitness of the fish. Statistical analyses revealed no significant decrease in size in terms of length and weight at test end compared to control values (Jonckheere-Terpstra Test, α=0.05; one-sided smaller). Thus, the NOEC based on the endpoint size in terms of length at test end was defined as ≥ 105.44 μg a.i./L (mean measured concentration).

Values were furthermore evaluated separately for males and females.

No impaired growth in terms of length and weight at test termination for males and females were observed in treated fish compared to controls.

Mean length of male fish was in the range of 2.9 to 3.2 cm. The mean length of females was in the range of 2.9 and 3.5 cm. The mean weight of males was between 0.223 g and 0.300 g, the mean weight of females was between 0.251 g and 0.476 g. Statistical analysis revealed no significant differences (Jonckheere-Terpstra Test, α=0.05, one-sided smaller). Thus, the NOEC for the endpoints related to growth was determined as ≥ 105.44 μg a.i./L.

Pseudo-specific growth rate

The pseudo-specific growth rate from 35 dpf until 63 dpf was calculated. No statistically significant differences were observed for controls and treatments (Jonckheere-Terpstra Test α=0.05, one-sided smaller). The pseudo-specific growth rate in controls was calculated to be 2.152. Under treatment conditions, pseudo-specific growth rates of 2.088 to 2.299 were calculated, with no concentration-response relationship.

Sex ratio

The sex ratio in the replicates was determined. Sex determination was performed macroscopically by inspection of the gonads and furthermore histopathologically in order to determine the sex of very immature and to verify the sex of the other individuals. The sex ratio (in % females) was determined to be between 46.3 % and 62.1 % females with no test item-related tendency. This ratio match the requirements of a sex ratio between 30 – 70 % females.

Compared to the control, no test item-related effect was observed (Jonckheere-Terpstra Test α=0.05, two-sided). Based on these data, the NOEC for the endpoint sex ratio was determined to be ≥ 105.44 μg a.i./L.

Vitellogenin content in blood plasma

Due to the early developmental stage of fish, VTG values were low for single individuals (irrespective of the sex). Values that were below the LOQ at the lowest dilution (1:50) were considered as zero.

No statistically significant differences between controls and treatments were determined for the VTG content female fish (Jonckheere-Terpstra Test, α=0.05, two-sided). For controls, mean values of 317.9 ng VTG/μg protein were measured, while treatments displayed VTG concentrations between 567.4 ng VTG/μg protein (1.19 μg a.i./L) and 991.2 ng VTG/μg protein (32.51 μg a.i./L).

For males, mean measured VTG values ranged between 0.06 ng VTG/μg protein (105.44 μg a.i./L) and 1.03 ng VTG/μg protein (3.26 μg a.i./L, with no statistically significant differences between controls and treatments (Jonckheere-Terpstra Test, α=0.05, two-sided).

Based on these data, the NOEC for the biomarker VTG content in blood plasma was determined to be ≥ 105.44 μg a.i./L.

Histopathology

Sex ratio/sexual development

The examined fish were assigned to 3 different categories: female, male or transition phase. The latter describes a juvenile, immature individual that is most probably a male, but still in the natural transition phase from juvenile ovary to testis. No significant change of the percentage of fish in transition phase could be observed.

Females

The average maturity index of females ranged between 2.7 (controls) and 4.3 (32.51 μg a.i./L) and showed a concentration-related increase, which was statistically significant at the two highest concentrations (32.51 μg a.i./L and 105.44 μg a.i./L; Jonckheere-Terpstra Test, p<0.05; two-sided). However, this effect was related to the overall increased size at higher concentrations, which is due to the lower number of individuals. Thus, the effect was considered as secondary effect and not related to any endocrine potential of the test item.

Histopathological lesions in the ovaries were not frequent or severe. The high percentage of females with egg debris at a concentration of 32.51 μg Preventol A6/L is due to the low number of only three females at a concentration of 32.51 μg preventol A 6/L, replicate 4. All of them showed egg debris.

Males

Testis maturity indices ranged between 2.3 (3.26 μg a.i./L) and 2.8 (11.32; 32.51, and 105.44 μg a.i./L) and showed no clear concentration-related changes.

Single immature oocytes were found in the testis tissue of some individuals (testis-ova). However, no concentration-related change in frequency or severity of this effect could be identified. Pathological alterations of testicular morphology were not observed.

Conclusion on histopathological results

Examination of the sex ratio of exposed fish showed that the chemical treatment did not have a strong effect on sexual differentiation. The slight feminization in the low exposure groups did not occur in the highest exposure groups. Additionally, the chemical exposure seemed to have a slightly stimulating effect on female gonad maturation, which, however, was statistically not significant. This effect is likely due to the lower number of individuals, which promoted faster growth and development.

Histopathological lesions in females (extended areas of egg debris and atresia, combined with inflammation) were rare, not very pronounced and not concentration-related. In males, no pathological lesions could be diagnosed. Except for the exposure with 32.51 μg a.i./L, all groups had a low percentage of male fish who were categorized as testis-ova. However, the occurrence did not change significantly, compared to the control.

Validity criteria fulfilled:
yes
Conclusions:
Preventol A6 caused in an OECD 234 study decreased post-hatch survival rates while there was no effect on endocrine-related endpoints. Thus, there is no indication that Preventol A6 is an endocrine disruptor. The NOEC was determined as 1.19 µg a.i./L (mean measured).
Executive summary:

An OECD 234 study on fish sexual development with Preventol A6 and the acitve ingredient (a.i.) diuron (purity ≥ 98.0 %) was conducted under flow-through conditions and documented via GLP. Zebrafish were exposed starting with fertilized eggs until 63 dpf with 1, 3.16, 10, 31.6 and 100 µg a.i./L (nominal concentration).

Chemical analysis with HPLC/MS-MS was performed to allow quantification of the a.i. with a LoD of 0.3 μg a.i./L. As few samples differed from the desired 80 - 120 % of the nominal concentration, it was decided to base the biological effects on mean measured concentrations (1.19 μg a.i./L; 3.26 μg a.i./L; 11.32 μg a.i./L; 32.51 μg a.i./L; 105.44 μg a.i./L).

Endpoints inspected were hatching rate, post-hatch survival at 35 dpf, length at 35 dpf, post-hatch survival at 63 dpf, length and weight at 63 dpf, pseudo-specific growth rate, sex ratio, Vtg content in blood plasma, and histopathology. There were no effect with endocrine-related endpoints (Vtg and sex ratio), as well as with growth in terms of length and weight. The NOEC for these endpoints was, thus, ≥ 105.44 µg a.i./L (mean measured). However, there was a significant effect on post-hatch survival at 35 dpf and 63 dpf which resulted in a NOEC of 1.19 µg a.i./L (mean measured).

In conclusion, Preventol A6, i.e. the active ingredient diuron, caused in an OECD 234 study decreased post-hatch survival rates while there was no effect on endocrine-related endpoints. There is no indication that diuron is an endocrine disruptor for wildlife. The NOEC was determined as 1.19 µg a.i./L (mean measured).

Description of key information

An OECD 234 study on fish sexual development with Preventol A6 and the acitve ingredient (a.i.) diuron (purity ≥ 98.0 %) was conducted under flow-through conditions and documented via GLP. Zebrafish were exposed starting with fertilized eggs until 63 dpf with 1, 3.16, 10, 31.6 and 100 µg a.i./L (nominal concentration).

Chemical analysis with HPLC/MS-MS was performed to allow quantification of the a.i. with a LoD of 0.3 μg a.i./L. As few samples differed from the desired 80 - 120 % of the nominal concentration, it was decided to base the biological effects on mean measured concentrations (1.19 μg a.i./L; 3.26 μg a.i./L; 11.32 μg a.i./L; 32.51 μg a.i./L; 105.44 μg a.i./L).

Endpoints inspected were hatching rate, post-hatch survival at 35 dpf, length at 35 dpf, post-hatch survival at 63 dpf, length and weight at 63 dpf, pseudo-specific growth rate, sex ratio, Vtg content in blood plasma, and histopathology. There were no effect with endocrine-related endpoints (Vtg and sex ratio), as well as with growth in terms of length and weight. The NOEC for these endpoints was, thus, ≥ 105.44 µg a.i./L (mean measured). However, there was a significant effect on post-hatch survival at 35 dpf and 63 dpf which resulted in a NOEC of 1.19 µg a.i./L (mean measured).

In conclusion, Preventol A6, i.e. the active ingredient diuron, caused in an OECD 234 study decreased post-hatch survival rates while there was no effect on endocrine-related endpoints. There is no indication that diuron is an endocrine disruptor for wildlife. The NOEC was determined as 1.19 µg a.i./L (mean measured).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
1.19 µg/L

Additional information

The NOEC was given as mean measured concentration of the active ingredient diuron.