Reaction mass of 2,6-bis(1-phenylethyl)phenol and 2,4,6-tris(1-phenylethyl)phenol

Long-term toxicity to fish

Administrative data

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

Study period:

20-02-2018 to 26-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

long-term toxicity to fish, other

Remarks:

Fish Sexual Development Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-02-2018 to 26-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 234 Fish Sexual Development Test

Version / remarks:

July 2011

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. QA statement)

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

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

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:

secondary sexual characteristics

Remarks:

sex ratio

Remarks on result:

other:

Remarks:

% females

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:

Remarks:

% undifferentiated

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

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:

Remarks:

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 (undiffererentiated) 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 three different categories: female, male, or undifferentiated. The category ‘undifferentiated’ described individuals, where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes [7]. The gonads of these animals were most probably arrested in the development into testis due to the treatment with the test item, and afterwards remained at an early ovary stage. Furthermore, it described fish in transition phase, with gonads consisting of undeveloped germ cells to oogonia exclusively. For these animals, it was not possible to confirm the sex. Individuals, which were assigned as ‘unidentified – no gonads’, were taken out of the calculation, as these individuals could not be sexed due to technical errors during cutting and slide preparation. Individuals compiled in this group did not represent a true separate sex category.
For determination of statistically significant differences, the percentage of mature males and females was considered. Furthermore, statistical differences with respect to the percentage
of undifferentiated fish were calculated.
In controls and in the four lower test concentrations, the mean value of %males ranged between 18.9 % (61.6 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration)) and 30.0 % (6.4 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration)). No mature males were found in any of the replicates of the highest test concentration (187.9 μg 4-(1-phenylethyl)-phenol/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. The percentage of females ranged between 36.2 % (187.9 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration)) and 60.2 % (control). No statistically significant differences was thus observed.
There was a concentration-related, statistically significant increase (p<0.05) in the mean value of undifferentiated fish from the concentration of 6.4 μg 4-(1-phenylethyl)-phenol/L. In controls, the mean value for undifferentiated fish was at 10.8 %, and increased concentration-depended to 63.8 % at the highest test level (187.9 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration)).
The NOEC for the endpoint sex ratio (%males) was defined as 61.8 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration). Regarding the number of undifferentated fish, the NOEC was defined as 2.1 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration). Single immature oocytes were found in the testis of some individuals (testis-ova; compare Annex A.6), which was defined as pathological alteration of the testis tissue. These individuals are however undoubtedly identified as males and not assigned to the category of ‘intersex’.

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. Fish which could not be determined histopathologically (unidentified – no gonads) 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. A statistically significant increase in mean VTG values at the highest treatment level (187.9 μg 4-(1-phenylethyl)-phenol/L) was also determined for undifferentiated fish. Based on the results for mature females and for undifferentiated fish, the NOEC for the biomarker VTG was determined at 61.8 μg/L (mean measured concentration).

Results with reference substance (positive control):

/

Biological effects during the Early Life Stage phase of the study

		Nominal concentration 4 -MSP [µg/L]
		Control	2.0	6.3	20.0	63.0	200.0
		Mean measured concentration 4-MSP [µg/L]
		Control	2.1	6.4	19.7	61.8	187.9
No of replicates		4	4	4	4	4	4
Post hatch survival, 21 dpf [%]	Mean	78.5	79.0	65.0	82.5	83.3	84.1
	SD	12.3	11.3	11.4	11.3	9.4	11.0
	RSD	15.6	14.3	17.5	13.8	11.3	13.0
Post hatch survival, 35 dpf [%]	Mean	78.5	76.5	65.0	81.7	80.8	80.0
	SD	12.3	12.5	11.4	11.1	8.3	11.8
	RSD	15.6	16.3	17.5	13.5	10.3	14.8
Length; 35 dpf [cm]	Mean	1.89	1.95	1.99	1.93	1.98	1.98
	SD	0.21	0.02	0.07	0.07	0.06	0.07
	RSD	11.1	0.8	3.4	3.4	3.0	3.8

SD = Standard deviation

RSD = Relative standard deviation

Biological effects at test termination

		Nominal concentration 4-MSP [µg/L]
		Control	2.0	6.3	20.0	63.0	200.0
		Mean measured concentration 4-MSP [µg/L]
		Control	2.1	6.4	19.7	61.8	187.9
No of replicates		4	4	4	4	4	4
Survival, 63 dpf [%]	Mean	77.7	76.5	64.2	80.0	80.8	79.1
	SD	11.6	12.5	10.3	11.5	8.3	12.8
	RSD	15.0	16.3	16.1	14.4	10.3	16.2
Length, all individuals; 63 dpf [cm]	Mean	3.1	3.2	3.2	3.1	3.2	3.2
	SD	0.2	0.0	0.1	0.1	0.1	0.1
	RSD	5.6	1.5	2.5	2.4	2.8	2.7
Weight, all individuals; 63 dpf [g]	Mean	0.333	0.336	0.352	0.317	0.328	0.321
	SD	0.060	0.026	0.027	0.021	0.029	0.033
	RSD	17.9	7.7	7.8	6.6	8.7	10.3
Length males; 63 dpf [cm]*^#	Mean	3.2	3.2	3.2	3.1	3.3	-
	SD	0.1	0.1	0.1	0.2	0.1	-
	RSD	2.1	1.8	2.2	5.2	3.4	-
Length females; 63 dpf [cm]*	Mean	3.2	3.2	3.3	3.3	3.2	3.3
	SD	0.2	0.1	0.1	0.1	0.1	0.1
	RSD	4.8	3.0	3.1	2.7	3.2	2.5
Weight males; 63 dpf [mg]*^#	Mean	0.306	0.308	0.311	0.283	0.329	-
	SD	0.027	0.013	0.023	0.036	0.027	-
	RSD	8.7	4.2	7.4	12.8	8.3	-
Weight females; 63 dpf [mg]*	Mean	0.382	0.374	0.406	0.368	0.352	0.373
	SD	0.066	0.043	0.037	0.038	0.033	0.041
	RSD	17.2	11.4	9.2	10.4	9.4	11.0
Sex ratio [% females]##	Mean	60.2	57.9	49.5	41.2	51.0	36.2
	SD	5.8	18.0	7.3	10.3	10.2	12.5
	RSD	9.6	31.1	14.8	24.9	19.9	34.4
Sex ratio [% males]##	Mean	28.9	28.2	30.0	22.4	18.9	0.0***
	SD	15.1	6.8	3.2	15.7	6.5	0.0
	RSD	52.4	24.0	10.5	70.2	34.5	-
Sex ratio [% undifferentiated]##	Mean	10.8	13.9	20.5	36.4**	30.1**	63.8**
	SD	10.7	13.1	4.8	11.2	14.3	12.5
	RSD	98.9	94.4	23.6	30.7	47.5	19.5

SD = Standard deviation; RSD = Relative standard deviation

*          Only mature fish with clearly identified sex (histopathology) were considered for evaluation. Fish which could not be determined histopathologically (unidentified) or undifferentiated fish were excluded from calculation.

**        Williams t-test, p<0.05; one-sided greater

*** Jonckheere-Terpstra test, p<0.05; one-side smaller

# No values could be obtained for the highest concentration as no mature male fish could be identified.

## Fish defined as ‘unidentified - no gonads’ were not considered for determination of the sex ratio

Vitellogenin content in blood plasma

		Nominal concentration 4-(1-phenylethyl)-phenol [µg/L]
		Control	2.0	6.3	20.0	63.0	200.0
		Mean measured concentration 4-(1-phenylethyl)-phenol [µg/L]
		Control	2.1	6.4	19.7	61.8	187.9
No of replicates		4	4	4	4	4	4
VTG / total protein [ng/µg]; females*	Mean	480.8	407.8	500.3	404.5	451.9	729.7**
	SD	204.0	82.2	65.5	163.4	181.2	307.4
	RSD	42.4	20.2	13.1	40.4	40.1	42.1
VTG / total protein [ng/µg]; males*#	Mean	0.08	0.39	0.77	0.04	0.07	-
	SD	0.02	0.61	1.04	0.02	0.08	-
	RSD	28.1	158.2	134.6	52.9	108.4	-

TG / total protein [ng/µg]; undifferentiated*	Mean	0.70	92.01	2.37	2.40	21.30	319.69***
	SD	0.91	183.11	2.43	2.83	25.88	129.31
	RSD	130.3	199.0	102.7	118.0	121.5	40.5

SD = Standard deviation

RSD = Relative standard deviation

Remark:Values below LOQ were considered as zero

* Fish which could not be determined histopathologically (unidentified – no gonads) were excluded from calculation.

** Williams t-test, p<0.05; one-sided greater

*** Jonckheere-Terpstra test, p<0.05; one-sided greater

# No values could be obtained for the highest concentration as no mature male fish could be identified.

Measured test item concentrations during the in-life phase of the study

Nominal concentration 4-MSP	Replicate	Measured concentration 4-MSP
		[µg/L] / vessel		[%] / vessel		[µg/L] / treatment		[%] / treatment
[µg/L]		Mean	SD	Mean	SD	Mean	SD	Mean	SD
Control	A	< LOQ	-	-	-	< LOQ	-	-	-
	B	< LOQ	-	-	-
	C	< LOQ	-	-	-
	D	< LOQ	-	-	-
2.0	A	2.3	0.2	116.7	8.5	2.1	0.2	105.7	9.5
	B	2.1	0.8	106.9	9.3
	C	2.0	0.8	105.8	13.2
	D	1.8	0.7	93.4	15.5
6.3	A	6.7	0.4	105.9	6.6	6.4	0.4	102.1	5.9
	B	5.9	0.7	93.3	10.7
	C	6.6	0.8	105.0	12.6
	D	6.6	1.2	104.2	19.0
20.0	A	20.6	4.7	110.1	15.3	19.7	0.9	104.3	6.0
	B	18.6	3.6	97.4	15.3
	C	20.2	4.2	108.4	10.0
	D	19.4	3.5	101.3	15.1
63.0	A	63.9	8.3	105.8	7.4	61.8	2.2	100.2	3.8
	B	58.8	9.4	97.4	11.2
	C	62.1	6.1	98.6	9.7
	D	62.3	7.0	98.9	11.2
200.0	A	181.0	26.4	86.2	14.5	187.9	14.4	92.0	9.4
	B	171.0	29.9	81.8	14.9
	C	198.8	33.4	99.4	16.7
	D	200.9	32.9	100.4	16.5

Remark: Due to inadequate dilution of the respective samples, few measured values were outside the applied calibration range.

This refers to the following samples:

- day 0, vessels 5/3 and 5/4
- day 21, vessel 3/1
- day 28, vessel 3/4
Nevertheless, a calculation based on extrapolation is considered possible and scientifically justified as the data obtained were in line with subsequent measurements. As a result, the actual standard deviation may be higher. However, an effect on the overall data variability could not be found.

Validity criteria fulfilled:

yes

Conclusions:

4-MSP displays a strong estrogenic mode of action.

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 undifferentiated fish ranged between 10.8 % (controls) and 63.8 % (187.9 μg 4-(1-phenylethyl)-phenol/L), with a monotonous concentration-response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg 4 -(1-phenylethyl)-phenol/L.

Regarding the number of undifferentiated fish, the NOEC was defined as 2.1 μg 4-(1 -phenylethyl)-phenol/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. A statistically significant increase in mean VTG values at the highest treatment level (187.9 μg 4-(1-phenylethyl)-phenol/L) was also determined for undifferentiated fish, from 0.70 ng VTG/μg protein in controls to 319.69 ng VTG/μg protein at the highest treatment level.

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

 

Conclusion

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 and undifferentiated fish.

Histopathological examination further indicated an increased percentage of undifferentiated fish 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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Reduced approach with only 3 test concentrations and a control, in 2 replicates each

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

p-(1-phenylethyl)phenol

EC Number:

217-864-1

EC Name:

p-(1-phenylethyl)phenol

Cas Number:

1988-89-2

Molecular formula:

C14H14O

IUPAC Name:

4-(1-phenylethyl)phenol

Test material form:

solid

Details on test material:

- Density: 1.074 g/cm3

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.

Sampling and analysis

Analytical monitoring:

no

Remarks:

No analytical monitoring was performed during the range finding pre-test.

Test solutions

Vehicle:

no

Test organisms

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 (main test).
- 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.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

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

Details on test conditions:

DETERMINATION OF PRE-TEST DOSE LEVELS
The OECD 234 guideline recommends that concentrations higher than 10% of the acute LC50 need not to be tested. Furthermore, the maximum test concentration should be 10% of the LC50 on the larval/juvenile life stage. Available data on the toxicity of a mixture of styrenated phenols documented an LC50 of 1.77 mg/L 4-MSP. However, no data on larval/juvenile toxicity were available. Therefore, the pre-test was based on the Fish Early-Life Stage test (OECD 210), with 2.0 mg/L as the highest test concentration.

TEST SYSTEM
- No. of organisms per vessel: 30 fertilized eggs were introduced at test start.
- No. of vessels per concentration (replicates): 2 replicates for each test concentration (i.e. 60 fertilized eggs per test concentration).
- No. of vessels per control (replicates): 2 replicates

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
- Post-hatch survival
- Size at day 21

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

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.

Results with reference substance (positive control):

/

Any other information on results incl. tables

		Nominal concentration 4-(1-phenylethyl)-phenol [mg/L]
		Control	0.02	0.2	2.0
No of replicates		2	2	2	2
Hatch, 4 dpf [%]	Mean	98.4	93.4	100.0	0.0
Hatch, 5 dpf [%]	Mean	100.0	100.0	100.0	100.0
Post hatch survival, 21 dpf [%]	Mean	81.7	93.3	88.3	0.0
Length, 21 dpf [cm]	Mean	1.03	1.04	1.05	-
Individual dry weight, 21 dpf [mg]	Mean	1.7	1.6	1.8	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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.

Executive summary:

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.