Phenol, mono- + distyrenated (MSP+DSP)

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Remarks:

extended to a Fish Sexual Development Test (FSDT)

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:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

extended study including juvenile state and sexual differentiation

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 Strasse 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 frozen on 15-01-2018 were thawed 26-04-2018 and analysed 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 µS/cm

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 (see also separate entry)
- 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 at 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:

35 d

Dose descriptor:

NOEC

Remarks:

highest concentration tested

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:

hatching success and rates, larval mortality and development

Key result

Duration:

63 d

Dose descriptor:

NOEC

Remarks:

highest concentration tested

Effect conc.:

> 187.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: juvenile growth: length and weight

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).
Furthermore, values were 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 (undifferentiated) 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.

Results with reference substance (positive control):

not included

Reported statistics and error estimates:

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.

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

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 immature 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.

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 did not produce any statistically significant adverse effects on the endpoints hatch, post-hatch survival, and growth. The NOEC was >0.188 mg/L, the highest concentration tested. On the other hand, 4-MSP showed estrogenic activity (for this endpoint, see separate entry under "6.1.8 Endocrine disrupter testing....").

Executive summary:

An extended Early-Life-Stage Fish Test 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 guidelines 210 and 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 which were not indicative of 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).

Endpoints indicative of endocrine-mediated effects were the sex ratio and the vitellogenin (VTG) concentration in blood plasma (see separate entry).

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. The biological effects were bsed on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L and 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 for males and females at 63 dpf when compared to controls.

Of note, for males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified.

 

Conclusion

There was no statistically significant effect of the test item 4-MSP on the endpoints hatch, post-hatch survival, and growth. Based on toxicological findings, the chronic NOEC for toxicity is set at >0.188 mg/L, corresponding to 0.2 mg/L (nominal).

Description of key information

For a constituent of phenol, styrenated, 4-(1-phenylethyl)phenol, data on long-term fish toxicity are available from an OECD 234 Fish Sexual Development Test. Based on fish early-life-stage (FELS) parameters, a NOEC of 0.188 mg/L was derived.

In a study according to OECD TG 204 using Japanese ricefish, a 14d-NOEC for phenol, styrenated was determined. In addition, the 14d-LC50 (3.8 mg/L) is reported.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Remarks:

highest concentration tested

Effect concentration:

0.188 mg/L

Additional information

Reporting for the OECD 204 study is very limited. Details on test performance and results are not available from the database. The substance tested was phenol, styrenated (CAS 61788-44-1). Composition of the test substance is not reported, but from the substance name under the Chemical Substance Control Law (mono (or di or tri)-(alpha-methylbenzyl)phenol) it can be concluded that the test substance is a mixture of mono-, di-, and tristyrenated phenol. Similarity with Novares LS 500 is considered to be adequate to use data from this test as substantial evidence for the characterisation of Novares LS 500.

The OECD 234 Fish Sexual Development Test (see IUCLID Section 6.6) was carried out with the test substance 4-(1-phenylethyl)phenol (4-MSP), a constituent of phenol, styrenated (Novares LS 500), under GLP conditions. Even though the concentration of the test substance in phenol, styrenated is only ca. 44 %, the test result of this test is considered to better represent the long-term fish toxicity of total phenol, styrenated, because the FELS test has been found to be much more sensitive than the older, out-of-date OECD 204 test (Fish, Prolonged Toxicity Test: 14-day Study). Therefore, the results of the new test with the test substance 4-(1-phenylethyl)phenol (4-MSP) is selected out of the available data to represent the long-term fish toxicity of phenol, styrenated.