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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 - Dose Range FInder
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 234 Fish Sexual Development Test
Deviations:
yes
Remarks:
Minor deviations in test temperature and mean measured concentrations. These deviations did not influence the overall validity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Germany GmbH, Varziner Strase 49, 47138 Duisburg (Germany)
- Lot/Batch n°: Lab Sample 6.9.2017
- Expiration date of the lot/batch: 31-12-2018
- Purity test date: 06-09-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material: room temperature, dry
- Storage stability of the test item in frozen samples: Spiked samples forzen on 15-01-2018 were thawed 26-04-2018 and analyzed alongside the t63 samples. The analysis of the thawed samples confirmed the storage stability.
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Justification: Danio rerio are selected in accordance with OECD 234 guideline.
- Common name: Zebrafish
- Origin of the used strain of zebrafish: West Aquarium GmbH, 37431 Bad Lauterberg, Germany.
- Source of fertilized eggs: Egss were obtained from individuals reared in the test facility (Fraunhofer Institute, Schmallenberg, Germany).
- Maximum age of parental fish: 2 years.
- Holding conditions of parental fish: temperature 26°C +/- 2°C; light-dark cycle 12h/12h; daily feeding ad libitum with TetraMin Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina). The broodstock is permanently visually checked 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.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Eggs were collected in a glass spawning-tray which was placed at the bottom of the holding vessels. The tray was covered with a stainless steel lattice to prevent adult fish from predating the eggs. An artificial substrate was attached to the lattice to stimulate spawning into the tray. The turn on of lighting (one neon lamp per vessel, light intensity approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel) induced mating and spawning of fish.
- 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.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
63 d
Hardness:
1.0 - 1.1 mmol/L
Test temperature:
27.0 +/- 2.0°C
pH:
7.74 - 8.02
Dissolved oxygen:
9.18 – 9.36 mg/L
98.3 – 103.0 %
Conductivity:
246 – 254
Nominal and measured concentrations:
Nominal concentrations range finding pre-test: 0.00, 0.02, 0.2 and 2.0 mg/L
Nominal concentrations main test: 0.0, 2.0, 6.3, 20.0, 63.0, 200.0 µg/L
Measured concentrations main test: < LOQ, 2.1, 6.4, 19.7, 61.8 and 187.9 µg/L
Details on test conditions:

TEST SYSTEM
- Test vessel: 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. 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.
- No. of organisms per vessel: 30 fertilized eggs were placed in the the 2 glass fry cages (15 in each fry cage) that are present in each vessel.
- No. of vessels per concentration (replicates): 4 replicates for each test concentration (i.e. 120 fertilized eggs per test concentration).
- No. of vessels per control (replicates): 4 replicates
- 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.
- Biomass loading rate: 1g fish /L maximum.
- Renewal rate of test solution (frequency/flow rate): 8 volumes per vessel per day. Flow through volumes were checked weekly and did not vary by more than 10%.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: drinking water, purified by activated charcoal filtration and aeration.
- Total Organic Carbon content: 0.3752 - 6.5240 mg/L
- Metals: < 0.278 µg/L Cd, < 0.543 µg/L Cr, < 1.012 µg/L Cu, < 0.760 - 4.35 µg/L Fe, < 0.366 µg/L Mn, < 3.16 µg/L Ni, < 3.28 µg/L Pb, < 0.879 - 1.23 µg/L Zn
- Chlorine: < 0.02 - 0.03 mg/L
- Nitrate: 11 - 13 mg/L
- Nitrite: < 0.005 mg/L
- Ammonium: < 0.01 mg/L
- Phosphate: 0.18 - 0.31 mg/L
- Calcium: 0.8 - 0.9 mmol/L
- Magnesium: 0.1 - 0.3 mmol/L
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Photoperiod: 12h light / 12h dark
- Light intensity: approx. 1000 lux, measured 5 cm above the water surface in the middle of the test vessel
- Aeration: no (oxygen concentration did not fall below 60%)
- Post-hatch feeding: When hatch was finished, i.e. from day 5 dpf on, larvae were fed twice daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany) and liquid rearing feed (Nobil fluid, JBL, Neuhofen, Germany). From day 14 (dpf) on, brine shrimp nauplii (Artemia salina) were added twice daily. From day 14 (dpf) on, ground TetraMin flakes were added once daily to the fish feed. From 14 dpf on, larvae were allowed to freely move to the main vessel in order to support undisturbed growth.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Hatching and survival: daily observations. On day 21 and 35, post-hatch survival counts are made by means of digital photography.
- Observations on abnormal appearance of behaviour: daily observations.
- Length determination: by digital photography on day 35 and on day 63.
- Weight determination: on day 63 (wet weight, blotted dry).
- Sex ratio determination: macroscopical determination by inspection of the gonads, and confirmation by histopathological examination.
- Determination of the Vitellogenin (VTG) concentration in the blood plasma by means of ELISA kits.

TEST CONCENTRATIONS
- Test concentration selection: based on pre-test (see below).
- Test concentrations: 2.0, 6.3, 20, 63 and 200 µg/L.
- Spacing factor for test concentrations: Square root of 10
- Justification for using less concentrations than requested by guideline:

RANGE-FINDING STUDY
- Range-finding test method: reduced test design similar to OECD 210, with 3 test concentrations and a control, in two replicates each. In each replicate, 30 eggs were introduced at the test start.
- Range-finding test duration: 21 days.
- Range-finding test concentrations: 2.0, 0.2 and 0.02 mg/L
- Results used to determine the conditions for the definitive study:
- Hatch: Hatch was delayed in the highest test concentration, which displayed no hatch at 4 dpf. Controls and the two other treatment levels already displayed nearly 100 % hatch at this time point. At 5 dpf, all replicated, including the highest treatment level, displayed 100 % hatch.
- Post-hatch survival: Mortality was observed at the highest treatment level, already at 6 to 7 dpf. At 21 dpf, all larvae were found dead at 2.0 mg/L, resulting in a post-hatch survival rate of 0 %. At the two other treatment levels, no difference of the post-hatch survival rate to controls was observed (81.7 % in controls compared to 93.3 % and 88.3 % at the lower and middle treatment level).
- Length and weight at 21 dpf: No negative effect of the test item was observed.
- Selection of concentrations for the main test: The pre-test demonstrated that a concentration of 2.0 mg/L resulted in 100% mortality. Thus, it was decided that a maximum test concentration of 10% of this test concentration is appropriate as highest test level.

SPECIFIC METHOD INFORMATION:
- Photography and image analysis: For fry counts and total length measurements, photographs were made using the digital camera: Canon Cybershot (Canon, Tokio, Japan). Digital image processing was performed by using UTHSCSA ImageTool Version 3.0 (University of Texas Health Science Center at San Antonio, USA). Fish were netted and placed in glass vessels with a low water level. This was placed in the photo device (light plate with additional illumination from above). After photographing, the fry were carefully re-introduced into the test vessel.
- 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 (5g/L). Plasma was separated from the blood via centrifugation (30 min; 5000 g; 4°C) and immediately stored at -80 °C until further analysis.
- Vitellogenin measurement: For determination of the vitellogenin levels, an enzyme-linked immunoabsorbant assay (ELISA) raised to zebrafish (Danio rerio) VTG (homologous ELISA kit, Biosense, Bergen, Norway) was used. The VTG analysis is based on a sandwich assay utilizing specific binding between antibodies and VTG. The wells of microtiter plates were coated with a specific capture antibody that binds to VTG in samples added to the wells. Unbound components were washed out, and a different VTG-specific antibody (detecting antibody) was added. Unbound detecting antibody was washed out, and an enzyme-labelled secondary antibody was added. After a last wash, the enzyme activity was determined by adding a substrate being metabolized to a colored product. The enzyme activity (color intensity) measured by a microplate reader is directly proportional to the concentration of VTG in the sample. The assay was calibrated using purified VTG from zebrafish as a standard. A blank control was study part in each assay. Furthermore a fortified blood plasma sample was prepared and measured on each plate. For this a male blood sample was spiked with a known amount of VTG standard. The ratio of expected concentration to the measured concentration was reported along with the results from each set of assays.
In order to minimize variability generated by the blood sampling methods (e.g. by taking up tissue liquid), the measured VTG concentrations were normalized for the blood plasma protein content, expressed as ng VTG/μg protein. Total protein was quantified by using the BCA Protein Assay Reagent Kit (Pierce, Rockford, USA). The method of the BCA Protein Assay combines the reduction of Cu2+ to Cu+ and allows a selective colorimetric detection of the cuprous cation (Cu+) using a reagent containing bicinchoninic acid. The colored reaction is formed by chelation of two molecules of BCA with one cuprous ion. This complex shows a strong absorbance at 562 nm which is almost linear with increasing protein concentration.

STATISTICAL CALCULATIONS:
For each endpoint, the NOEC and LOEC were determined. All statistics were calculated using ToxRat® Professional 3.2, respectively the most actual version of this program.
For NOEC / LOEC-determination, quantal data were arcsine-transformed prior to analysis. No Observed Effect Concentrations (NOEC) were calculated, using ANOVA, followed by Williams t-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.
Reference substance (positive control):
no
Key result
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
61.8 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
secondary sexual characteristics
Remarks:
sex ratio
Remarks on result:
other: % males
Key result
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
> 187.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: length and weight
Key result
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
2.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
secondary sexual characteristics
Remarks:
sex ratio
Remarks on result:
other: % females / stage 0
Key result
Duration:
63 d
Dose descriptor:
NOEC
Effect conc.:
61.8 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: VTG biomarker
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
> 187.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Early Life Stage observations
Remarks on result:
other: no significant effect on post-survival hatch or larval growth. observed
Details on results:
EARLY LIFE STAGE ENDPOINTS (UP TO DAY 35):

Hatching rate:
First larvae hatched at 3 dpf across all treatment levels. Hatch was completed at 5 to 6 dpf in all replicates, with no difference between treatments. Only few eggs were found coagulated during this period. 93.3 to 100.0 % hatch was observed in all replicates. Thus, the validity criterion on hatch (> 80 %) was fulfilled.

Post-hatch survival:
Survival was recorded daily on a visual basis and at 21 and 35 dpf by photographic counting. Between 6 dpf and 13 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. This life phase is known to be very sensitive, leading to increased mortality even under non-exposed conditions. Surviving larvae did not display any signs of disease.
At 21 dpf, the post-hatch survival rate in controls reached a mean value of 78.5 %. At 35 dpf, post-hatch survival in control vessels was also at 78.5 % and thus met the validity criterion for survival in controls of ≥ 70 %. The post-hatch survival rate in treatments was between 65.0 % (6.4 μg/L (mean measured concentration) and 81.7 % (19.7 μg/L (mean measured concentration). One replicate (replicate B) in the second treatment level (6.4 μgl/L (mean measured concentration)) displayed a decreased post-hatch survival rate. The mean post- hatch survival rate was however not significantly different from controls.
Statistical analyses of post-hatch survival at 35 dpf revealed no significant differences between control and treatments. Thus, the NOEC for the endpoint post-hatch survival during ELS was defined at ≥ 187.9 μg/L (mean measured concentration).

Length at day 35:
At 35 dpf, larval growth in terms of length was determined. In this study, mean length of 1.89 cm was observed in controls. The mean length of larvae in the treatment conditions were found in the range of 1.93 cm (19.7 μg/L) and 1.99 cm (6.4 μg/L). Statistical analyses revealed no significant decrease in size compared to control values. Thus, the NOEC based on the endpoint size in terms of length after the ELS was defined as ≥ 187.9 μg/L (mean measured concentration).

ENDPOINTS AT TEST TERMINATION (DAY 63):

Survival:
One juvenile fish in the controls and two juvenile fish at mean measured concentrations of 187.9 μg/L were found dead during the juvenile phase. No other signs of toxicity were observed. Statistical analyses of post-hatch survival at 63 dpf revealed no significant differences between control and treatments. The NOEC for the endpoint post-hatch survival at test end was defined at ≥ 187.9 μg/L (mean measured concentration). Thus, no test item-related effect on post-hatch survival between 35 dpf and test end was observed.

Length and weight:
At 63 dpf, growth in terms of length was determined. Juvenile fish in the control displayed a mean measured length of 3.1 cm. The mean length of fish under treatment conditions varied between 3.1 cm and 3.2 cm, with no concentration-response relationship. The mean measured weight of fish in controls was measured at 0.333 g, the mean weight of fish under treatment conditions varied between 0.317 g and 0.352 g, again with no concentration- response relationship.
Statistical analyses revealed no significant decrease in size in terms of length and weight at test end compared to control values. Thus, the NOEC based on the endpoint size in terms of length at test end was defined as ≥ 187.9 μg/L (mean measured concentration).
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. Fish which could not be determined histopathologically (unidentified) or immature fish (female/stage 0; transition phase, see below for details) were excluded from calculation. For males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified. No differences in terms of length and weight at test end were observed for males and females, respectively. Females displayed mean lengths between 3.2 cm and 3.3 cm, and mean weights between 0.352 g and 0.406 g. Males displayed mean lengths between 3.1 cm and 3.3 cm and mean weights between 0.283 g and 0.329 g.

Sex ratio:
The sex ratio was determined based on histopathological examinations of the gonads. The examined fish were assigned to five different categories: female, male, female/stage 0, transition phase or unidentified. Female/stage 0 described individuals, where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes. The gonads of these animals were most probably arrested in the development into testis due to the treatment with the test item, and afterwards developed towards female. Animals in transition phase described animals with gonads consisting of undeveloped germ cells to oogonia exclusively. For these animals, it was not possible to confirm the sex. The category ‘unidentified animals’ described fish where no gonads were visible on the histological slide, even after recutting.
For determination of statistically significant differences, the percentage of mature males was considered. Furthermore, statistical differences with respect to the percentage of females/stage 0 were calculated.
In controls and in the four lower test concentration, the mean value of %males ranged between 18.6 % (61.6 μg/L (mean measured concentration)) and 30.0 % (6.4 μg /L (mean measured concentration)). No mature males were found in any of the replicates of the highest test concentration (187.9 μg/L (mean measured concentration)). A statistically significant difference (p<0.05) in the sex ratio in terms of %males was thus observed for the highest test concentration.
There was a concentration-related, statistically significant increase (p<0.05) in the mean value of %females/stage 0 from the concentration of 6.4 μg/L. The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration). Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

Vitellogenin content in blood plasma:
The VTG content in the blood plasma of all individuals was determined. However, only the values of mature males and females were considered for analysis. Fish which could not be determined histopathologically (unidentified) or immature fish (female/stage 0; transition phase) were excluded from calculation.
A statistically significant increase in mean VTG values was reported in females, only at the highest test concentration (187.9 μg/L). There were no significant differences in VTG contents for males at any concentration level. Of note, VTG content could not be determined at the highest concentration as no mature males were present. Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg/L (mean measured concentration).
Validity criteria fulfilled:
yes
Conclusions:
There was no statistically significant effect of the test item 4-MSP on the endpoints hatch, post-hatch survival, and growth. Results however indicated a feminizing effect of 4-MSP on the test species zebrafish (Danio rerio). Determination of the sex ratio indicated a statistically significant decrease of the number of males in the highest test concentration. Furthermore, the mean values for the biomarker VTG indicated a statistically significant increase of the VTG concentration in the blood plasma of females. Histopathological examination further indicated an increased percentage of immature individuals (statistically significant increase of the number of females/stage 0) already at a concentration of 6.4 μg/L (mean measured concentration). Interpretation of the obtained results (an increase of the VTG content in females as well as the absence of males in the highest test concentration) suggests a strong estrogenic mode of action for the test substance. This interpretation follows the OECD TG 234. Based on these endocrine-related endpoints, the following NOEC was determined (based on the measure %males, corresponding to an increase of the measured VTG concentration in female blood plasma): NOEC of 61.8 μg/L 4-MSP (mean measured concentration), corresponding to NOEC of 63.0 μg/L 4-MSP (nominal concentration).
Executive summary:

A fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1 -phenylethyl)-phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/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 and which were not indicative for endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post fertilization (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). An endpoint that is indicative for endocrine-mediated effects and was determined during the study was the sex ratio. 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, a biomarker also indicative for endocrine modes of action. Furthermore, a histopathological examination of the fish gonads was performed.

 

Chemical analysis

The concentrations of the 4-MSP were assessed by chemical analysis using GC-MS. The LOQ was set to 0.75 μg 4-MSP/L. Mean concentrations per treatment of the 4-MSP during the course of the study were between 92.0 % and 105.7 % of the nominal concentration of the test item. As some samples differed from the desired 80 – 120 % of the nominal values, it was decided to base the biological effects on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;
 187.9 μg/L).

 

Non-endocrine apical endpoints:
 Early life stage

· Hatching rate: Hatch was completed at 5 to 6 dpf in all replicates, with no difference between treatments.

· Post-hatch survival at 35 dpf: There was no test-item related effect on post-hatch survival at 35 dpf when compared to controls. 

· Length at 35 dpf: There was no test-item related effect on length at 35 dpf when compared to controls.

 

Non-endocrine apical endpoints: Test termination

· Post-hatch survival at 63 dpf: There was no test-item related effect on post-hatch survival at 63 dpf when compared to controls.

· Length and weight at 63 dpf: There was no test-item related effect on length and weight at 63 dpf when compared to controls. No differences in terms of length and weight at test end were observed for males and females, respectively. Of note, for males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified (details in section “Sex ratio” below).

 

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item related effect on sex ratio in terms of %males was reported at 187.9 μg/L. Indeed, no mature males were found in any of the replicates of the highest test concentration whereas the mean value of %males ranged between 18.6 % and 30.0 % in controls and the four lower test concentrations. The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration).

The mean value of %females/stage 0 (individuals where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes) ranged between 8.9 % (controls) and 52.5 % (187.9 μg/L), with a monotonous concentration- response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg/L.

Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

 

Endocrine related endpoints and biomarkers: Vitellogenin content in blood plasma

When compared to controls, a statistically significant test-item related increase in vitellogenin content was reported at 187.9 μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg /L (mean measured concentration).

Description of key information

A fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1 -phenylethyl)-phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/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 and which were not indicative for endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post fertilization (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). An endpoint that is indicative for endocrine-mediated effects and was determined during the study was the sex ratio. 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, a biomarker also indicative for endocrine modes of action. Furthermore, a histopathological examination of the fish gonads was performed.

 

Chemical analysis

The concentrations of the 4-MSP were assessed by chemical analysis using GC-MS. The LOQ was set to 0.75 μg 4-MSP/L. Mean concentrations per treatment of the 4-MSP during the course of the study were between 92.0 % and 105.7 % of the nominal concentration of the test item. As some samples differed from the desired 80 – 120 % of the nominal values, it was decided to base the biological effects on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;
 187.9 μg/L).

 

Non-endocrine apical endpoints:
 Early life stage

· Hatching rate: Hatch was completed at 5 to 6 dpf in all replicates, with no difference between treatments.

· Post-hatch survival at 35 dpf: There was no test-item related effect on post-hatch survival at 35 dpf when compared to controls. 

· Length at 35 dpf: There was no test-item related effect on length at 35 dpf when compared to controls.

 

Non-endocrine apical endpoints: Test termination

· Post-hatch survival at 63 dpf: There was no test-item related effect on post-hatch survival at 63 dpf when compared to controls.

· Length and weight at 63 dpf: There was no test-item related effect on length and weight at 63 dpf when compared to controls. No differences in terms of length and weight at test end were observed for males and females, respectively. Of note, for males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified (details in section “Sex ratio” below).

 

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item related effect on sex ratio in terms of %males was reported at 187.9 μg/L. Indeed, no mature males were found in any of the replicates of the highest test concentration whereas the mean value of %males ranged between 18.6 % and 30.0 % in controls and the four lower test concentrations. The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration).

The mean value of %females/stage 0 (individuals where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes) ranged between 8.9 % (controls) and 52.5 % (187.9 μg/L), with a monotonous concentration- response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg/L.

Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

 

Endocrine related endpoints and biomarkers: Vitellogenin content in blood plasma

When compared to controls, a statistically significant test-item related increase in vitellogenin content was reported at 187.9 μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg /L (mean measured concentration).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.2 mg/L

Additional information

Various experimental studies of the test chemical were reviewed for long term toxicity to fish endpoint which are summarized as below:

 

In an experimental key study, a fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1 -phenylethyl)-phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/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 and which were not indicative for endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post fertilization (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). An endpoint that is indicative for endocrine-mediated effects and was determined during the study was the sex ratio. 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, a biomarker also indicative for endocrine modes of action. Furthermore, a histopathological examination of the fish gonads was performed.

 

Chemical analysis

The concentrations of the 4-MSP were assessed by chemical analysis using GC-MS. The LOQ was set to 0.75 μg 4-MSP/L. Mean concentrations per treatment of the 4-MSP during the course of the study were between 92.0 % and 105.7 % of the nominal concentration of the test item. As some samples differed from the desired 80 – 120 % of the nominal values, it was decided to base the biological effects on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;
 187.9 μg/L).

 

Non-endocrine apical endpoints:
 Early life stage

· Hatching rate: Hatch was completed at 5 to 6 dpf in all replicates, with no difference between treatments.

· Post-hatch survival at 35 dpf: There was no test-item related effect on post-hatch survival at 35 dpf when compared to controls. 

· Length at 35 dpf: There was no test-item related effect on length at 35 dpf when compared to controls.

 

Non-endocrine apical endpoints: Test termination

· Post-hatch survival at 63 dpf: There was no test-item related effect on post-hatch survival at 63 dpf when compared to controls.

· Length and weight at 63 dpf: There was no test-item related effect on length and weight at 63 dpf when compared to controls. No differences in terms of length and weight at test end were observed for males and females, respectively. Of note, for males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified (details in section “Sex ratio” below).

 

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item related effect on sex ratio in terms of %males was reported at 187.9 μg/L. Indeed, no mature males were found in any of the replicates of the highest test concentration whereas the mean value of %males ranged between 18.6 % and 30.0 % in controls and the four lower test concentrations. The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration).

The mean value of %females/stage 0 (individuals where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes) ranged between 8.9 % (controls) and 52.5 % (187.9 μg/L), with a monotonous concentration- response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg/L.

Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

 

Endocrine related endpoints and biomarkers: Vitellogenin content in blood plasma

When compared to controls, a statistically significant test-item related increase in vitellogenin content was reported at 187.9 μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg /L (mean measured concentration).

 

In a supporting study, a range finder pre-test was performed prior to a fish sexual development test (FSDT) with zebrafish (Danio rerio). The range finder pre-test design was based on the OECD 210 Fish Early-Life Stage (FELS) test, but with a reduced test setup consisting of 3 concentrations plus a control, in two replicates each. The tested concentrations were 0, 0.02, 0.2 and 2.0 mg/L, and the following parameters were monitored: hatch, post-hatch survival and growth. Hatch was found to be delayed in the highest test concentration. Hatch mainly occurred on day 4 post fertilization in the lower dose groups, but only on day 5 in the highest dose group. Mortality started to occur in the highest dose group at day 6 -7 post fertilization. At study termination, all larvae were found dead. At the two other treatment levels, the post-hatch survival rate was similar as that of the control. At study termination, no effect on the growth or dry weight of the surviving larvae of the lower dose groups was observed.At 2.0 mg/L (nominal), 100% mortality was observed. At lower dose levels (0.2 and 0.02 mg/L, nominal), no effects on mortality or growth were observed.On the basis of effect on mortality of the test organism, the 21 d NOEC value was determined to be 0.2 mg/l.

 

On the basis of the above results of long term toxicity to fish study as per OECD TG 234 of 4-(1 -phenylethyl)-phenol (4-MSP), the 21 d NOEC value was determined to be in the range of 0.0618 to > 0.18 mg/l, respectively. Based on the OECD TG 210 study of 2,4,6-tris(1-phenylethyl)phenol, the 21 d NOEC value was determined to be 0.2 mg/l.