Benzotriazole

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish: other

Remarks:

fish sexual development

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 16, 2018 – Dec 18, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 234 (Fish Sexual Development Test)

Version / remarks:

28.07.2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Unknown impurities

IUPAC Name:

Unknown impurities

Constituent 1

Reference substance name:

Benzotriazole

EC Number:

202-394-1

EC Name:

Benzotriazole

Cas Number:

95-14-7

Molecular formula:

C6H5N3

IUPAC Name:

benzotriazole

Test material form:

solid: granular

Sampling and analysis

Analytical monitoring:

yes

Remarks:

Besides the parent substance also the metabolites 4-hydroxybenzotriazol and 5-hydroxybenzotriazol were monitored in water and fish tissue. Methods were validated according SANCO/3029/99 rev. 4.

Details on sampling:

At test start, after 7 and 14 days samples were taken from all aquaria. From day 21 on water samples were taken weekly from alternating lines of the test system. Samples from the additional vessels were taken accordingly. Samples from the additional vessels were taken from all four aquaria at test start. During exposure the samples were taken weekly from alternating lines of the flow through system. An aliquot was taken and used for analytical measurement. The remaining amount of the samples was immediately stored at ≤ -18 °C.
Water samples for the analysis of both metabolites were taken in parallel to the samples for the parent substance. The samples were stored deep frozen (< -18 °C) until analysis. For the analysis of Benzotriazole and both metabolites in fish tissue, the fish were humanely killed and the biological parameter were determined after the exposure. The fish were snap-frozen at - 80 °C and afterwards stored deep frozen (< -18 °C) until further analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: dilution in test medium, multiple steps necessary to prepare stock solutions for flow through application, test contrations were adjusted by flow rate of stock solution and dilution water
- Eluate: -
- Differential loading: -
- Controls: -
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): -
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): -
- Test concentration separation factor: √10
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
- Other relevant information:

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain origin: West Aquarium GmbH, 37431 Bad Lauterberg, Germany
- Source: Test facility bred, IME, Auf dem Aberg 1, Schmallenberg
- Life stage: fertilized eggs, obtained from individuals reared in the laboratory
- Age at study initiation (mean and range, SD): -
- Length at study initiation (length definition, mean, range and SD): -
- Weight at study initiation (mean and range, SD): -
- Method of breeding:
Parental fish for egg production were hold in aquaria with a total volume of 150 L. Holding water was of the same quality as used in the test (purified drinking water, see below). The maximum age for parental fish was 2 years. Stock density was approximately 1 g/L volume. Holding temperature was 26 °C ± 2 °C. Light/dark cycle was 12 h/12 h. Flow through rate was adjusted to reach approx. one total exchange of water per day. Fish were fed daily ad libitum with TetraMin® Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina).
The broodstock was visually checked every day for mortality, illness, parasites or abnormal behavior. No prophylactic treatment of fish took place. Only healthy fish without diseases and abnormalities were used as parental fish for the production of fertilized eggs.

- Pre-exposure reproductive information

ACCLIMATION
- Acclimation period: not applicable
- Acclimation conditions (same as test or not):not applicable
- Type and amount of food during acclimation:not applicable
- Feeding frequency during acclimation: not applicable
- Health during acclimation (any mortality observed): not applicable

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable


FEEDING DURING TEST
- Food type: TetraMin® Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina)
- Amount: ad libitum
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs):
- Method of collection of fertilised eggs: glass spawning-tray covered with a stainless steel lattice
- Subsequent handling of eggs: The collected eggs were transferred from the spawning-tray into a sieve, rinsed with clean water in order to remove any debris and then put into glass dishes. Fertilized eggs (microscopic determination of > four cell stage, i.e. early blastula stage) were transferred by means of a widened and de-burred pipette tip into the test chambers. Time from spawning until transferring into the test solutions was kept as short as possible and no later than 12 h after fertilization.
- Subsequent handling of juvenile fish:

POST-HATCH FEEDING
From day 7 on (dpf = days post fertilization), larvae were fed once daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany). From day 14 (dpf) on, brine shrimp nauplii (Artemia salina) were added once daily. From the same date on, ground TetraMin flakes were added once daily to the fish feed.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

63 d

Test conditions

Hardness:

total: 1.2-1.4 mmol/L

Test temperature:

27.0 °C ± 2.0 °C

pH:

6.26-7.24

Dissolved oxygen:

87-117 %

Conductivity:

295 – 323 µS/cm

Nominal and measured concentrations:

nominal: 0.10 0.32, 1.00, 3.20, 10.0 mg/L (main vessels); 1.00, 10.0 mg/L (add. vessels)
measured: 0.104, 0.331, 1.07, 3.34, 11.0 mg/L (main vessels); 1.11, 10.9 mg/L (add. vessels)

Details on test conditions:

TEST SYSTEM (schemata in background material)
- Test vessel: aquaria 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.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 28 L total volume, approx. 25 L fill volume
- Aeration: not necessary, continuous water exchange
- Type of flow-through (e.g. peristaltic or proportional diluter): dosage system with membrane pumps and mixing chamber
- Renewal rate of test solution (frequency/flow rate): 5 volumes per day
- No. of organisms per vessel: 2 x 15 fertilized and randomized eggs
- No. of organisms per mL or well: -
- No. of vessels per concentration (replicates): 4 ( + 2 at medium test concentration and + 2 at highest test concentration for examination of metabolites)
- No. of vessels per control (replicates): 4
- Vehicle control performed: no
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate: -
- Stocking density: -
- No. of independent and valid run (solutions and spawn): -


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water (filtration with activated charcoal and aeration)
- Total organic carbon: 0.3985 – 0.6134 mg/L (DOC)
- Particulate matter: n/a
- Metals (cadmium, chrome, copper, iron, manganese, nickel, lead and zinc): < 15 µg/L (sum parameter)
- Pesticides: n/a
- Chlorine: 0.02 mg/L
- Alkalinity: 1.8 – 2.2 mmol/L
- Ca/mg ratio: 1:2
- Culture medium different from test medium: no
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: yes, (37 % HCl) via dosage system
- Photoperiod: 12/12 light/dark
- Light intensity: approx. 1000 lumen

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
3 dpf: Time to hatch, Hatching success
21 dpf: post-hatch survival
35 dpf: post-hatch survival, length
63 dpf: survival, length and weight, sex ratio, gonad histopathology, VTG content, liver and kidney histopathology

TEST CONCENTRATIONS
- Spacing factor for test concentrations:√10
- Justification for using less concentrations than requested by guideline:

RANGE FINDING STUDY (test was performed following the basic requirements of OECD guideline 210)
- Test concentrations: 0.1, 1.0, 10 mg/L
- Results used to determine the conditions for the definitive study: No effects on survival were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

1.07 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival

Effect concentrations 2

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 3

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

all fish

Effect concentrations 4

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

all fish

Effect concentrations 5

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

males

Effect concentrations 6

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

females

Effect concentrations 7

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

undiff

Effect concentrations 8

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

males

Effect concentrations 9

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

females

Effect concentrations 10

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

undiff

Effect concentrations 11

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Remarks:

%females/%males

Effect concentrations 12

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Remarks:

%females

Effect concentrations 13

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Remarks:

%males

Effect concentrations 14

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Remarks:

%undiff

Effect concentrations 15

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks:

females

Effect concentrations 16

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks:

males

Effect concentrations 17

Duration:

63 d

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks:

undiff

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: complete hatch observed (100 %), post hatch survival: varied between 83.3 % (mean measured concentration of 11.0 mg benzotriazole/L) and 92.5 % (mean measured concentration of 0.10 mg benzotriazole/L)
- Overall mortality/survival: Three days before test termination (60 dpf), unexpected mortality was observed in two vessels of the control and of the first treatment level, each (vessel A and B for each treatment). In total, 21 fish in controls and 20 fish at a treatment level of 0.10 mg benzotriazole/L (mean measured) were found dead. The remaining fish did not show any signs of disease.
- Days to hatch or time to release of young: hatch started on 2 dpf and completed at 4 dpf
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: complete hatch observed
- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): -
- Observations on body length and weight of young and/or exposed parents at one or more time periods: information on length and weight in attachment "Biological effects"
- Number of healthy fish at end of test: control: (94/120), 0.1 mg/L (91/120), 0.33 mg/L (107/120), 1.07 mg/L (113/120), 3.34 mg/L (100/120), 11.0 mg/L (100/120)
- Type of and number with morphological abnormalities: -
- Type of and number with behavioural abnormalities: -
- Type and number of developmental / reproductive effects: -
- Type and number of abnormal pigmentation: -
- Type and magnitude of hormonal changes:-
- Detailed data on spawning, egg numbers, fertility, and fecundity: -
- Other biological observations: -
- Effect concentrations exceeding solubility of substance in test medium: no
- Concentrations that produce lethal or other effects: [describe and attach graph showing effects with respect to time]: post hatch survival rate at 35 dpf shows significant difference at 11 mg/L.
- Cumulative mortality at each concentration and for each recommended observation time if possible: 4 dpf: no mortality; 21 dpf: 5 fish (control), 9 fish (0.1 mg/L), 11 fish (0.33 mg/L), 5 fish (1.07 mg/L), 16 fish (3.34 mg/L), 18 fish (11 mg/L); 35 dpf: 5 fish (control), 9 fish (0.1 mg/L), 12 fish (0.33 mg/L), 7 fish (1.07 mg/L), 19 fish (3.34 mg/L), 20 fish (11 mg/L); 63 dpf: 26 fish (control), 29 fish (0.10 mg/L), 13 fish (0.33 mg/L), 7 fish (1.07 mg/L), 20 fish (3.34 mg/L), 20 fish (11 mg/L)
- Mortality in the controls: on 60 dpf 21 fish were found dead. No signs of disease were observed
- Incidents in the course of the test which might have influenced the results: -
- Fish weights (individual and mean values): mean values in attachment "biological effects"

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

For each endpoint, the NOEC and LOEC were determined. All statistics were calculated using ToxRat® Professional 3.3.
For NOEC / LOEC-determination, quantal data were arcsin-transformed prior to analysis. No Observed Effect Concentrations (NOEC) were calculated, using ANOVA, followed by Dunnetts t-test, Williams test, or respective non-parametric approaches (e.g. Jonckheere-Terpsta test.
Details of all statistical analyses are reported, including exact p-values for all statistical comparisons. Prior to use of parametric procedures, results of tests of normality and homogeneity of variance were considered. Failure to confirm assumptions of normality and homogeneity of variance resulted in the use of a suitable non-parametric test for the data involved. Results of all tests for normality and homogeneity of variance are reported along with the results of the parametric or non-parametric tests.
If the test results show a concentration-response relationship, with a maximum effect size of at least 50 %, the data were analyzed by regression to determine the ECx-values including the 95 % confidence interval using Probit-analysis assuming log-normal distribution of the values.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see attached document 'validity criteria'

Conclusions:

Statistical evaluation revealed a reduced post-hatch survival rate during the early life stages (determined at 35 dpf) for the two highest concentrations of benzotriazole. Thus, for the endpoint post-hatch survival at 35 dpf, the NOEC was defined as 1.07 mg benzotriazole/L (mean measured concentration). However, this effect was considered as not relevant, as the post-hatch survival rate in the highest treatment reached more than 80 % (83.3 %) and was thus well above the validity criterion set for controls (75 %).

No negative effects were observed for the endpoint growth at test termination, as fish were evaluated as being larger under treatment conditions compared to controls.



Evaluation of the effects of benzotriazole on the sex ratio of exposed fish did not show a concentration-response relationship. Even though an increased number of undifferentiated fish under treatment conditions as well as a decreased number of males was observed, these effects did not follow a linear trend, i.e., no concentration-response relationship was observed. Statistically significant effects were only observed especially for the lowest treatment. No trend to a shift of the sex ratio towards males or females was observed. The slight decrease of the number of females under treatment conditions is not significant. It could be attributed to the undifferentiated state of the majority of fish, which was even more evident in treatments compared to controls. An indication that the sex ratio is not shifted is given if only the sex ratio of males and females is compared, which showed similar levels between controls and treatments and no statistically significant effect.



Thus, based on the present data, no endocrine effect was observed by the treatment with benzotriazole up to a concentration of 11.0 mg/L.



NOEC ≥ 11.0 mg benzotriazole/L (mean measured concentration),

corresponding to

NOEC ≥ 10.0 mg benzotriazole/L (nominal concentration).

Executive summary:

A fish sexual development test (FSDT) with zebrafish (Danio rerio), sponsored by Connect Chemicals GmbH, was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME). The objective of this study was the assessment of effects of continuous exposure to the test item on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234 [5]. The study was conducted with nominal concentrations of 0.10, 0.32, 1.00, 3.20, 10.0 mg benzotriazole/L in four replicates each under flow through conditions. An untreated control was run in parallel. Exposure was started with 30 fertilised eggs per test vessel and replicate. Endpoints that were determined included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 pf and when groups were terminated (day 63 pf), fish were digitally photographed. Fish lengths were determined by evaluating photographs using electronically supported analysis. Single wet weights (blotted dry) were determined on day 63 pf (test end).

Sex ratios were determined macroscopically by inspection of the gonads and by histopathological verification. Blood samples of all fish were taken and measured for the vitellogenin (VTG) concentration. Furthermore, a histopathological examination of the fish gonads was performed [7].

Chemical analysis

Mean concentrations per treatment of the benzotriazole during the course of the study were between 56.8 % (nominal concentration of 1.00 mg benzotriazole/L) and 141.6 % (nominal concentration of 10.0 mg benzotriazole/L) of the nominal concentration of the test item. As the mean measured concentrations thus differed from the desired 80 – 120 % of nominal values, it was decided to base the biological effects on mean measured concentrations (0.10, 0.33, 1.07, 3.34, 11.0 mg/L).

Biological effects

Early life stage

Hatching rate

First larvae started to hatch at 2 dpf across all treatment levels. Hatch was completed at 4 dpf in all replicates, with no difference between treatments. Complete hatch was observed in all replicates.

Post-hatch survival at 35 dpf

Survival rates under treatment conditions at 35 dpf varied between 83.3 % (mean measured concentration of 11.0 mg benzotriazole/L) and 92.5 % (mean measured concentration of 0.10 mg benzotriazole/L). Statistical analyses of post-hatch survival at 35 dpf revealed significant differences between control and the two highest treatment conditions (Williams Test, p<0.05; one-sided smaller), with a monotonous concentration-response relationship. Thus, the NOEC for the endpoint post-hatch survival during ELS was defined at 1.07 mg benzotriazole/L (mean measured concentration).

Total length at 35 dpf

At 35 dpf, larval growth in terms of length was determined. In this study, mean length of 1.88 cm was observed in controls. The mean length of larvae in the treatment conditions were found in the range of 1.79 cm (11.0 mg/L; mean measured) and 1.88 cm (3.34 mg/L, mean measured).

Statistical analyses revealed no monotonous concentration-response relationship. The lowest and the highest test concentration were statistically significantly smaller than controls (Dunnett’s t-test; p<0.05; one-sides smaller). However, differences were smaller than 5 % and were thus considered as not relevant.

Test termination

Post-hatch survival at 63 dpf

Three days before test termination (60 dpf), unexpected mortality was observed in two vessels of the control and of the first treatment level, each (vessel A and B for each treatment). In total, 21 fish in controls and 20 fish at a treatment level of 0.10 mg benzotriazole/L (mean measured) were found dead. The remaining fish did not show any signs of disease.

Due to this mortality, the survival rate in controls was at 78.3 % at test termination. Survival rates under treatment conditions were between 75. 8% (0.10 mg benzotriazole/L; mean measured) and 94.2 % (1.07 mg benzotriazole/L; mean measured). Statistical analyses of post-hatch survival at 63 dpf could not be performed due to the increased mortality in controls 3 days before test termination.

Total length and wet weight at 63 dpf

At 63 dpf, growth in terms of length was determined. Juvenile fish in the control were smaller than fish under treatment conditions. As increasing size is not considered as adverse, no statistical analysis was performed.

Values were furthermore evaluated separately for males and females. Only mature fish with histopathologically clearly identified sex were considered for evaluation of the length and weight at test end. Immature fish (undifferentiated) were evaluated separately.

Control females displayed a mean length of 2.9 cm, and a mean weight of 0.242 g. Females under treatment conditions displayed a mean length between 2.9 cm in the four lower test concentrations and 3.0 cm at the highest treatment level. The mean weight ranged between 0.244 g (0.10 mg/L and 1.07 mg/L; mean measured) and 0.277 g (11.0 mg/L; mean measured).

Control males displayed a mean length of 2.7 cm, and a mean weight of 0.201 g. Males under treatment conditions displayed a mean length between 2.6 cm (0.10 mg/L; mean measured) and 2.9 cm at the highest treatment level. The mean weight ranged between 0.177 g (0.10 mg/L; mean measured) and 0.233 g (11.0 mg/L; mean measured).

Undifferentiated fish in controls displayed a mean length of 2.7 cm, and a mean weight of 0.207 g. Undifferentiated fish under treatment conditions displayed a mean length between 2.8 cm ( 0.10 mg/L; 0.33 mg/L; 1.07 mg/L; 11.0 mg/L; mean measured) and 2.9 cm at 3.34 mg/L. The mean weight ranged between 0.216 g (0.10 mg/L; mean measured) and 0.242 g (3.34 mg/L; mean measured).

Thus, fish under treatment conditions were slightly larger than fish in controls. As an increased growth was not considered as negative, no statistical analysis was performed, as no decrease was observed.

Sex ratio

The obtained histological data confirmed a trend to a decreased number of females in treatments compared to the control, while the number of undifferentiated fish increased, however not in a concentration-dependent manner. In controls, the mean value for females was at 47.8 % of all fish. In treatments, the mean value for females ranged between 20.4 % (1.07 mg benzotriazole/L; mean measured) and 32.6 % (11.0 mg benzotriazole/L; mean measured), with no concentration-response relationship. Statistical analyses revealed that there was no statistically significant effect on the % females (Dunnett’s t-test; two-sided; α=0.05).

The number of undifferentiated fish increased from a mean value of 26.1 % in controls to mean values of 64.2 % (0.10 mg benzotriazole/L; mean measured) and 49.8 % (11.0 mg benzotriazole/L; mean measured). Similar to the endpoint % of females, no concentration-response relationship was observed for the endpoint % of undifferentiated fish. However, this increase was statistically significant at concentrations of 0.10 mg benzotrialzole/L (mean measured) and 1.07 mg benzotriazole/L (mean measured; Dunnett’s t-test; two-sided; p<0.05).

The number of males slightly decreased, from a mean value of 26.1 % in controls to mean values between 6.7 % (0.10 mg benzotriazole/L; mean measured) and 17.6 % (11.0 mg benzotriazole/L; mean measured). The decrease in the relative value of males however also did not follow a linear trend. A statistically significant difference was found for effects on the number of males. At the first treatment level, a statistically significant decrease was observed (Dunnett’s t-test; two-sided; p<0.05).

Vitellogenin content in blood plasma

The VTG content in the blood plasma of all individuals was determined. The values of mature males and females, as well as values of undifferentiated fish were considered for analysis. Due to the young age of the fish and the undeveloped state, the VTG levels were also low for the females. No concentration-related increase or decrease of VTG levels was observed for females, males and undifferentiated fish. Thus, no statistically significant differences between control and treatment levels were observed for any of the evaluated sex categories (females; males: Dunnett’s t-test; two-sided; α=0.05; undiff: Bonferroni-Holm Median test; two-sided; α=0.05).