Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 October 2014 to 20 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP. Testing conducted in accordance with ECHA Decision number CCH-D-0000003873-68-05/F.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See Any other information

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None specified

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken in triplicate from the test concentration and singular samples were taken from the control.

Sampling:
Frequency: At the start and the end of one interval of 72 hours (nominal days 0 and 3) and one interval of 48 hours (nominal days 8 and 10). After this day no more samples were taken.
Volume: A sample volume of 20 ml was taken and spiked into a separation funnel containing 20 ml dichloromethane.
Storage: Not applicable, samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

The batch of LOWINOX® CPL tested was a UVCB and its appearance was described as a white to cream coloured powder. The substance was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test substance.

All test concentrations were prepared separately and started with loading rates of 1.0, 10 and 100 mg/l for the range-finding test and with a loading rate of 1.0 mg/l for the Early Life Stage (ELS) test. Two days of magnetic stirring at room temperature in the dark was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were filtered through a 0.45 µm membrane filter (Whatman) where after the clear and colourless Water Accommodated Fractions (WAFs) were used as the test concentrations.

Test organisms (species):

Pimephales promelas

Details on test organisms:

Test system
Species: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque.
Reason for selection: This system has been selected as an internationally accepted species.
Source: In house culture.

Holding of the brood stock
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l

Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, temperature was measured before transferring the parental fish to the breeding system.
Water quality parameters: Will be kept within the optimum limits for the respective fish species.
Ratio male/female: 1:2
Spawning tank: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands).
Time of fertilisation: Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

34 d

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Total hardness ranged between 214 and 268 mg calcium carbonate per litre. This was according to the value described in the protocol, i.e. >140 mg/L as CaCO3.

Test temperature:

The average temperature in the control and the test concentration varied between 22.8 and 25.8 °C. The temperature continuously measured in a temperature control vessel (nominal days 0-6) or in one of the replicates of the control (nominal days 6-34) ranged between 23.2 and 26.5 °C. This was not within the range described in the Protocol: 25 ± 1.5 °C.

pH:

The pH varied between 7.2 and 8.0 during the test period, which was within the range described in the Protocol (6.0-8.5).

Dissolved oxygen:

Oxygen concentrations varied between 5.2 and 10 mg/l during the test period, which is >60% of oxygen saturation at 25°C as prescribed by the Protocol.

Nominal and measured concentrations:

Measured concentrations
Concentrations in the test samples measured until day 10 of exposure were below or around the concentration of the lowest calibration solution, i.e. below 0.5 µg/l.
No more samples were taken and analyzed during the remainder of the test period based on the results of the samples taken until day 10 which, showed that the actual test concentration was either below or around the concentration of the lowest calibration solution. The effect parameters were therefore based on the loading rate rather than on the measured test concentration.

Details on test conditions:

Test procedure and conditions
Test type: Semi-static
Frequency of renewal: Three times a week
Test duration: 34 days
Introduction egg: Before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation).
Test vessels: Embryonic phase: Petri-dishes.
Early larval phase: stainless steel vessels of 1.7 litre, middle larval phase: all-glass vessels of 3 litres, late larval phase: all-glass vessels of 5 litres.
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Experimental design: The experiment (nominal day 0) started with 80 fresh and healthy fertilised fathead minnow eggs per test group. The fertilised eggs were randomly distributed and divided equally over four Petri-dishes. Each Petri-dish contained 20 eggs in 50 mL test medium and these dishes were incubated under gentle continuous shaking. On days 6, 24 and 27 the hatched larvae were transferred to increasingly larger vessels/volumes.
Light period: 16 h photo-period daily from 5 am until 9 pm; between 452 and 635 lux.
Feeding:
Embryonic phase: no feeding.
Newly hatched larvae: pelleted fish food (75 µm) and Brine shrimp Nauplii 24 hours old.
Juvenile stage: Brine shrimp Nauplii 24 or 48-hours old.
Food is supplied ad libitum.
Euthanasia: At the end of the test the surviving larvae were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Measurements and recordings
Organisms
Stage of embryonic development: Daily, from the beginning of the exposure
Hatching and survival: Daily, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording.
Criteria for death
Eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance;
Embryos: Absence of body movement and /or absence of heartbeat;
Larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.
Abnormal appearance: Daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and typical swimming behaviour.
Body weight: At the end of the test, all surviving fish were weighed on a replicate basis (blotted dry weight).
Body length: At the end of the test, all surviving fish were individually measured.

Test media
Oxygen, pH and temperature: At the start and the end of the test and at each renewal in each test group. In the freshly prepared solutions measurements were performed before exposing the organisms and in the old solutions after pooling of the replicates per group. In addition, temperature was recorded continuously.
Hardness: At the first and the last renewal interval in fresh and old media from each test group.

Reference substance (positive control):

no

Duration:

34 d

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

other: embryonic survival, larval survival and larval growth

Duration:

34 d

Dose descriptor:

LOELR

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

other: embryonic survival, larval survival and larval growth

Details on results:

Range-finding test
Hatching of embryo’s started at nominal day 3 and was finished at nominal day 5. Overall survival of fertilised eggs in the control was >70% until hatching was complete (90%). No significant effects on mortality of fish embryos and juvenile fish were observed at any of the test concentrations during the 7-day range-finding test.

All test conditions were maintained within the limits prescribed by the protocol.

Samples taken from the WAFs prepared at 10 and 100 mg/l were analysed but the analytical method used proved to be insufficient. It was however obvious that the actual concentrations in the WAFs were low, i.e. <0.01 mg/l.

After the performance of the range-finding test, the analytical method needed to be re-developed in order to measure lower test concentrations. It was decided to perform the ELS test with only one test concentration, i.e. a WAF prepared at a loading rate of 1.0 mg/l, since WAFs prepared at higher test concentrations would only introduce the risk of having undissolved test material in the test vessels. Preparation of WAFs below 1.0 mg/l is technically not feasible.

ELS test
Measured concentrations
Concentrations in the test samples measured until day 10 of exposure were below or around the concentration of the lowest calibration solution, i.e. below 0.5 µg/l.
No more samples were taken and analyzed during the remainder of the test period based on the results of the samples taken until day 10 which, showed that the actual test concentration was either below or around the concentration of the lowest calibration solution. The effect parameters were therefore based on the loading rate rather than on the measured test concentration.

Embryonic survival, development and hatching
Hatching of embryos started on the fourth day of exposure and was finished on the seventh day. Mean survival of the embryos in the control group was 96% and a similar percentage was reached in the test concentration (90%). Hatching in the test concentration started on the same day as in the control. Embryonic survival in the test concentrations was not statistically different from the control.

Larval survival and development
The mean post-hatch larval survival was 84% for the control. A similar percentage was reached for the test concentration (90%). Larval survival in the test concentration was not statistically different from the control. Some visible sub-lethal effects, i.e. malformation, no or less developed swim bladder, were recorded in the control.

Effects on larval growth
Both body length and body weight in the test concentrations were not statistically different from the control.

Experimental conditions
The pH varied between 7.2 and 8.0 during the test period, which was within the range described in the Protocol (6.0-8.5).

Oxygen concentrations varied between 5.2 and 10 mg/l during the test period, which is >60% of oxygen saturation at 25 °C as prescribed by the Protocol.

The average temperature in the control and the test concentration varied between 22.8 and 25.8 °C. The temperature continuously measured in a temperature control vessel (nominal days 0-6) or in one of the replicates of the control (nominal days 6-34) ranged between 23.2 and 26.5 °C. This was not within the range described in the Protocol: 25 ± 1.5 °C.

Total hardness ranged between 214 and 268 mg calcium carbonate per litre. This was according to the value described in the protocol, i.e. >140 mg/L as CaCO3.

T(D)OC measurement of a sample taken from the ISO-medium showed a value of 1.99 mg C/l, which was according to Protocol (<2 mg C/l).

Results with reference substance (positive control):

Reference substances not required for the study.

Reported statistics and error estimates:

STATISTICAL ANALYSIS ON EMBRYONIC SURVIVAL
Threshold Concentrations (NOELR) for Hatchability at 7 d
Chi² 2x2 Table Test with Bonferroni Correction

STATISTICAL ANALYSIS ON LARVAL SURVIVAL
Threshold Concentrations (NOELR) for Post-hatch survival at 34 d
Chi² 2x2 Table Test with Bonferroni Correction

STATISTICAL ANALYSIS ON BODY LENGTH
Shapiro-Wilk´s Test on Normal Distribution
Levene´s Test on Variance Homogeneity (with Residuals)
STUDENT-t test for Homogeneous Variances

STATISTICAL ANALYSIS ON BODY WEIGHT
Shapiro-Wilk´s Test on Normal Distribution
Levene´s Test on Variance Homogeneity (with Residuals)
STUDENT-t test for Homogeneous Variances

Survival of fish embryos and larvae during the range-finding test

LOWINOX® CPL WAF prepared at the given loading rate (mg/l)	No. of eggs	Number of survival							Total survival (%)
	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
Control	10	9	9	9	9	8	8	6	60
	10	10	10	10	10	10	10	7	70
1.0	10	10	10	10	10	10	10	10	100
	10	10	10	10	10	10	10	10	100
10	10	9	9	9	9	9	9	9	90
	10	9	9	9	9	9	9	9	90
100	10	9	9	9	9	9	9	9	90
	10	8	8	8	8	8	8	8	80

 

Embryonic survival and hatching success during the ELS test

LOWINOX® CPL WAF prepared at the given loading rate (mg/l)	Rep.^1	93 h		118 h		145 h		165 h		% Hatch Success
		No. Hatch	% Hatch	No. Hatch	% Hatch	No. Hatch	% Hatch	No. Hatch	% Hatch
Control	A	4	20	16	80	20	100	20	100	96
	B	7	35	16	80	19	95	19	95
	C^2	5	24	14	67	30	95	21	100
	D	7	35	17	85	18	90	18	90
1.0	A	7	35	15	75	17	85	17	85	90
	B	8	40	17	85	18	90	18	90
	C	7	35	16	80	18	90	18	90
	D	3	15	14	70	19	95	19	95

¹Twenty embryos were added to each vessel at the start of the test

²An additional embryo was found after two days of exposure

 

Effects on fish larvae of the control and the test concentration

Group (mg/l)

Vessel code

Day

Day

Day

Day

Day

Day

Day

Day

Day

Day

Day

Day

Day

5

6

7

8

9

10

11-15

16-17

18

19

20-21

22-24

25-34

Control

A

1p

1p

B

1h

1h

2z

2bz

1bz, 1s

1b

1s

1s

1s

C

2h

2h

1b,4z

1b,4z

1pbz,3bz

3b

3b

2s,1bs

3s

2s,1s

D

1m

1z

1.0

A

1y

1h

B

C

D

 

Legend of effect codes:

b	Bended corps/tail
p	Less pigmented
y	Malformed yolk sack
h	Cardiac edema
s	Small
m	Body malformed
z	No or less developed swim bladder

 

Individual body length (mm) of surviving larvae at the end of the test period

Code for larvae	Control				Loading rate 1.0 (mg/l)
	A	B	C	D	A	B	C	D
1	21.0	13.0	20.0	23.5	25.0	21.0	24.0	22.5
2	23.0	24.0	24.0	23.0	25.0	23.5	24.5	25.0
3	26.5	24.5	25.0	25.0	24.0	23.5	20.5	23.0
4	25.5	21.5	22.5	22.0	23.0	23.5	24.0	25.0
5	24.0	25.0	26.0	24.0	24.5	24.0	24.5	21.0
6	24.0	19.0	25.0	20.0	22.5	25.0	23.5	21.5
7	24.5	25.0	25.0	23.5	25.0	23.0	23.5	25.0
8	22.0	24.5	25.0	25.0	25.0	24.5	24.0	21.0
9	23.5	25.0	12.5	24.0	25.0	24.5	22.5	25.0
10	22.5	29.0	25.0	24.0	24.0	24.5	23.0	24.0
11	23.5	19.0	27.0	24.5	25.0	24.5	22.5	24.5
12	25.0	22.5	22.0	25.0	22.5	24.5	25.0	23.5
13	23.5	25.0	24.0	23.0		21.5	25.0	21.0
14	24.0	21.0	23.0	23.0		24.5	24.0	23.5
15	24.5	24.5	22.5	22.0		23.0	23.0	23.0
16	22.5	25.0	12.5	24.0		25.0	21.0	25.0
17			19.0			23.0	23.0	21.5
18							24.5	24.0
19
20
Minimum	21.0	13.0	12.5	20.0	22.5	21.0	20.5	21.0
Maximum	26.5	29.0	27.0	25.0	25.0	25.0	25.0	25.0
Average	23.7	23.0	22.4	23.5	24.2	23.7	23.4	23.3
SD	1.37	3.67	4.23	1.32	1.01	1.15	1.25	1.54
CV	6%	16%	9%	6%	4%	5%	5%	7%
n	16	16	17	16	12	17	18	18

SD=standard deviation, CV=coefficient of variation, n=number of surviving larvae

 

Individual body weight (mg wet weight) of surviving larvae at the end of the test period

Parameter	Control				Loading rate 1.0 (mg/l)
	A	B	C	D	A	B	C	D
All larvae	1877.2	1853.1	1758.5	1759.9	1719.8	1980.8	2032.1	2016.6
Mean	117.3	115.8	103.4	110.0	143.3	116.5	112.9	112.0
Mean per conc.	111.6				121.2
SD	6.3				14.9
CV	6%				12%
n	16	16	17	16	12	17	18	18

SD=standard deviation, CV=coefficient of variation, n=number of surviving larvae

Validity criteria fulfilled:

yes

Conclusions:

Testing conducted in accordance with ECHA Decision number CCH-D-0000003873-68-05/F

According to the integrated testing strategy, the Daphnia study was to be conducted first. If based on the results of the long-term Daphnia study and the application of a relevant assessment factor, no risks are observed (PEC/PNEC<1), no long-term fish testing may need to be conducted. However, if a risk is indicated, the long-term fish study needs to be conducted.

As effects were noted within the Daphnia study, and refinement of the PNEC was required, this test was conducted subsequently in order to allow for accurate risk assessment.

The study assessed the possible lethal and sub-lethal effects of LOWINOX® CPL during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions:

LOWINOX® CPL did not induce any statistically significant effects on embryonic survival at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for embryonic survival were 1.0 and >1.0 mg/l, respectively;
LOWINOX® CPL did not induce any statistically significant effects on larval survival at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for larval survival were 1.0 and >1.0 mg/l, respectively;
LOWINOX® CPL did not induce any statistically significant effects on larval growth at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for larval growth were 1.0 and >1.0 mg/l, respectively.

Executive summary:

Testing conducted in accordance with ECHA Decision number CCH-D-0000003873-68-05/F

According to the integrated testing strategy, the Daphnia study was to be conducted first. If based on the results of the long-term Daphnia study and the application of a relevant assessment factor, no risks are observed (PEC/PNEC<1), no long-term fish testing may need to be conducted. However, if a risk is indicated, the long-term fish study needs to be conducted.

As effects were noted within the Daphnia study, and refinement of the PNEC was required, this test was conducted subsequently in order to allow for accurate risk assessment.

Fish early-life stage toxicity test with LOWINOX® CPL (semi-static).

 

The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals: Guideline No. 210, 2013. In addition, the procedures were designed to meet the test methods and validity criteria of the EPA Ecological Effects Test Guidelines, 'Public Draft', EPA 712-C 96-121, 1996 and the OECD series on testing and assessment number 23, 2000.

 

The batch of LOWINOX® CPL tested was a UVCB and its appearance was described as a white to cream coloured powder. The substance was not completely soluble in test medium at the loading rates initially prepared.

 

A 34-day early life stage (ELS) test was performed based on the results of a preceding 7-day range-finding test. Preparation of the test solution started with a loading rate of 1.0 mg/l. Two days of magnetic stirring at room temperature in the dark was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a 0.45 µm membrane filter where after the clear and colourless Water Accommodated Fraction (WAF) was used as the test concentration.

 

The ELS test was performed under semi-static conditions with renewal of test solutions three times per week. The test was performed with four replicates of a control group and four replicates for the WAF. Both test groups contained 20 embryos per replicate. The test started by placing the embryos in Petri-dishes. On days 6, 24 and 27 hatched larvae and/or juvenile fish were transferred to larger vessels/volumes. During the embryonic and larval phases, the fish were observed for effects on development, appearance and swimming behaviour. At the end of the test, the surviving fish were measured and weighed.

 

During the test, samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of two renewal intervals. Concentrations in the test samples measured until day 10 of exposure were below or around the concentration of the lowest calibration solution, i.e. below 0.5 µg/l.

 

No more samples were taken and analyzed during the remainder of the test period based on the results of the samples taken until day 10 which, showed that the actual test concentration was either below or around the concentration of the lowest calibration solution. The effect parameters were therefore based on the loading rate rather than on the measured test concentration.

 

The study generally met the acceptability criteria prescribed by the protocol and was considered valid.

 

The results of the ELS test led to the following conclusions:

 

LOWINOX® CPL did not induce any statistically significant effects on embryonic survival at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the No Observed Effect Loading Rate (NOELR) and the Lowest Observed Effect Loading Rate (LOELR) for embryonic survival were 1.0 and >1.0 mg/l, respectively;

LOWINOX® CPL did not induce any statistically significant effects on larval survival at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for larval survival were 1.0 and >1.0 mg/l, respectively;

LOWINOX® CPL did not induce any statistically significant effects on larval growth at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for larval growth were 1.0 and >1.0 mg/l, respectively.

Description of key information

Long term toxicity to fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1 mg/L

Additional information

Testing conducted in accordance with ECHA Decision number CCH-D-0000003873-68-05/F

According to the integrated testing strategy, the Daphnia study was to be conducted first. If based on the results of the long-term Daphnia study and the application of a relevant assessment factor, no risks are observed (PEC/PNEC<1), no long-term fish testing may need to be conducted. However, if a risk is indicated, the long-term fish study needs to be conducted.

As effects were noted within the Daphnia study (although these are doubtful), and refinement of the PNEC was required, this test was conducted subsequently in order to allow for accurate risk assessment.

The study assessed the possible lethal and sub-lethal effects of LOWINOX® CPL during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions:

LOWINOX® CPL did not induce any statistically significant effects on embryonic survival at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for embryonic survival were 1.0 and >1.0 mg/l, respectively;

LOWINOX® CPL did not induce any statistically significant effects on larval survival at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for larval survival were 1.0 and >1.0 mg/l, respectively;

LOWINOX® CPL did not induce any statistically significant effects on larval growth at a concentration obtained in a WAF prepared at a loading rate of 1.0 mg/l. Hence, the NOELR and LOELR for larval growth were 1.0 and >1.0 mg/l, respectively.