1,4-phenylene bis((4-hydroxyphenyl)methanone)

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:

November 15, 2018 - July 9, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Long-term toxicity testing to fish is not a standard requirement according to Annex VIII. Since 1,3-Bis (4-hydroxy benzoyl) benzene is poorly water soluble (ca. 0.1 mg/L), a long-term toxicity study on fish was performed instead of a short-term toxicity study to fish. It should however been noted that this study was performed as part of an ongoing PreManufacture Notification ('PMN') in the US. For this reason no testing proposal for the performance of this study has been submitted.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

December 14, 2000

Deviations:

no

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: MMHBB-002/18
- Expiration date of the lot/batch: 01-APR-2021
- Purity test date: 09-APR-2018

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for analysis were taken from all test concentrations and both control groups. In addition, samples for analysis were taken from the stock solutions in DMSO prepared at Day -8 and at Day -3. These stocks were stored in a refrigerator for 7 and for 2 days, respectively, before analysis.
- Sampling frequency: One day before the start of exposure to check the functioning of the system (samples were taken from the test concentrations only). At the start, after 7, 14, 21, 28 and 32 days of exposure. In addition, a sample was taken on Day 22 from 0.10 mg/L after
a relatively low measured concentration on Day 21.
- Sampling volume: 1.0 mL from one replicate of each test group changing systematically amongst replicates.
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION OF STOCK SOLUTIONS
Stock solutions of 1.0 mg/mL were prepared in dimethylsulphoxide (DMSO; Merck, Darmstadt, Germany) three times a week. Lower stock solutions, i.e. 0.046, 0.10, 0.22 and 0.46 mg/mL were prepared by diluting the highest stock with DMSO. All stock solutions were clear and colourless.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name ans strain: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque.
- Source: In house culture.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- 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 (SDS 400, 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.

POST-HATCH FEEDING
Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)
Feeding: Embryonic phase: no feeding. Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48-hours old. Food was supplied ad libitum.

Test type:

flow-through

Water media type:

freshwater

Remarks:

ISO medium

Limit test:

no

Total exposure duration:

32 d

Hardness:

Total hardness was between 179 and 196 mg calcium carbonate per litre.

Test temperature:

23.7 - 24.9°C (test vessels)
23.2 - 24.4°C (control vessels)

pH:

7.0-7.6

Dissolved oxygen:

6.8 - 9.3 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0.0046, 0.010, 0.022, 0.046 and 0.10 mg/L
Measured concentrations (arithemetic means): 0.0044, 0.011, 0.026, 0.054 and 0.091 mg/L

Details on test conditions:

TEST SYSTEM
- Dosing system: Exact volumes of the test item stock solutions in DMSO were dosed with syringes via a computer-controlled system consisting of six dispensers (Gilson). The dosed volumes of the stock entered a mixing flask separately from the dilution water supply (adjusted ISOmedium). The dilution water was supplied applying flow-meters at a constant rate of 5 litres per hour.
In the mixing flask the dosed volume and the dilution water were mixed under continuous stirring. Peristaltic pumps, set at a rate of 1 litre per hour, were used to divide the contents of the mixing vessels continuously and equally over four replicate test vessels containing the
embryos/fish larvae/juvenile fish. The flow meters were calibrated before the start of the exposure and weekly thereafter. The whole system was checked twice daily on working days and once per day during weekends.
- Test type: Flow-through, with continuous renewal of test media
- Test vessel: stainless steel vessels (~1.7L)
- Aeration: no.
- Introduction of embryos: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation).
- No. of embryos per vessel: 20 embryos.
- No. of vessels per concentration (replicates): 4.
- No. of vessels per control (replicates): 4.
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM
- Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.

VEHICLE CONTROL PERFORMED: yes, test medium without test item but with 0.1 mL DMSO/L

OTHER TEST CONDITIONS
- Light period: 16h photo-period daily, between 464 - 823 lux.
- Feeding:
o Embryonic phase: no feeding;
o Larvae and juvenile fish: brine shrimp Nauplii 24 or 48-hours old (supplied ad libitum).

EFFECT PARAMETERS MEASURED
- Stage of embryonic development: at the beginning of exposure from at least a representative sample of eggs.
- 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.
- Abnormal appearance/behavior: daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and atypical 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, individual lengths from the surviving fish were measured.

RANGE-FINDING STUDY
- Test groups included 1.0, 10 and 100% of a 0.45 μm filtered solution prepared at 1.0 mg/L. A blank control group was also included. Each concentration consisted of two replicates containing a total of twenty eggs (ten each). The total test period was seven days. Test
conditions were held as similar as possible to those applied in the final test.

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Details on results:

EMBRYONIC SURVIVAL AND HATCHING
The two control groups (blank and solvent control) and were compared for hatchability and combined for statistical analysis since no significant difference was found between the control groups. The overall survival of embryos at the end of hatching was 96% in the pooled control, which complies with the requirements of the guideline (i.e. at least 70% of the embryos in the control should hatch). Hatching success was 95-100% in concentrations up to and including the highest average exposure concentration of 0.10 mg/L and was therefore not statistically different from the pooled control. Hatching of embryos started and ended between Day 4 and Day 7 of exposure for the control groups and all test concentrations. At Day 5 of exposure two embryos with cardiac oedema and other effects were observed in one replicate of the 0.010 mg/L concentration.

LARVAL SURVIVAL AND DEVELOPMENT
The two control groups (blank and solvent control) were compared for post-hatch survival and combined for statistical analysis since no significant difference was found between the control groups. The mean post-hatch larval survival was 94% in the pooled control. The validity criterion for post-hatch survival of at least 75% was met. The survival rates in the 1,3-Bis (4-hydroxy benzoyl) benzene treated groups was not dose related and ranged between 88 and 100%.
Statistical analyses indicated that larval survival was not statistically different from the pooled control at test concentrations up to and including 0.10 mg/L. A number of effects were observed in this study. Statistical analysis was not performed on abnormalities, but based on the observations made during the test it appears that the severity and incidence of abnormalities was not dose related.

EFFECTS ON LARVAL GROWTH
Because no statistically significant difference was observed between control treatments, the blank and solvent control groups were pooled. Mean body weight of the fish at the end of the test was 79 mg in the pooled control. Reduction of mean body weight in the 1,3-Bis (4-hydroxy benzoyl) benzene treated groups was not dose related and ranged between 6 and 11%. Statistical analyses indicated that body weight was not statistically different from the pooled control at test concentrations up to and including 0.10 mg/L. Mean body length of the fish at the end of the test was 21 mm in the pooled control. Reduction of mean body weight in the 1,3-Bis (4-hydroxy benzoyl) benzene treated groups was not dose related and ranged between 0 and 4%. A statistically significant reduction of length was observed at the lowest test concentration of 0.0046 mg/L, while all higher concentrations up to and including 0.10 mg/L were not statistically different from the pooled control. Reduction of mean body length in the lowest exposure concentration was caused by one fish with a body length of 12 mm, while all other fish had body length ranging between 17 and 24 mm. This, and the fact that no statistically significant reduction was observed at any of the higher concentrations, was reason to omit this result and state that body length was not different from the pooled control at test concentrations up to and including 0.10 mg/L.

Reported statistics and error estimates:

All statistical analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany). The following statistical procedures were used to determine the NOEC/LOEC for embryonic and larval survival, body weight and length.
Comparison of control treatments:
- Hatchability and Larval survival: Fisher`s Exact Binomial Test (alpha=0.05, two-sided)
- Weight and length: STUDENT-t test for Homogeneous Variances (alpha=0.05, two-sided) after check for normality of distribution and homogeneity of variances.
Because no statistically significant differences were detected for all parameters, the controls were pooled.
NOEC/LOEC for hatchability (embryonic survival):
- Step-down Cochran-Armitage Test Procedure (α=0.05, one-sided greater) after qualitative trend analysis by contrasts (monotonicity of concentration/response).
NOEC/LOEC for larval survival:
-Chi²-2 x 2 table test with Bonferroni Correction (α=0.05, one-sided greater) qualitative trend analysis by contrasts (monotonicity of concentration/response).

NOEC/LOEC for body weight and body length:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
- Dunnett’s Multiple t-test Procedure (α=0.05, one-sided smaller) after trend analysis by contrasts (monotonicity of concentration/response).

Table 1: Target, Measured and Average Concentrations

Test item conc. (mg/L)	Measured concentration (mg/L)						Average exposure conc. (mg/L)
	Day 0	Day 7	Day 14	Day 21	Day 28	Day 32
0.0046	0.0047	0.0045	0.0037	0.0048	0.0048	0.0041	0.0044 (96%)
0.010	0.0114	0.0094	0.0105	0.0128	0.0112	0.0105	0.011 (110%)
0.022	0.0263	0.0237	0.0262	0.0298	0.0222	0.0261	0.026 (117%)
0.046	0.0503	0.0432	0.0531	0.0589	0.0623	0.0574	0.054 (118%)
0.10	0.0914	0.105	0.0974	0.0391	0.113	0.0977	0.091 (91%)

Table 2: Percentage of Embryonic Survival at the End of Hatching

Treatment (mg/L)	Total Introduced	Hatched	Not Hatched	% Hatched
Pooled Control	160	154	6	96
0.0046	80	76	4	95
0.010	80	77	3	96
0.022	80	80	0	100
0.046	80	76	4	95
0.10	80	77	3	96

Table 3: Post-Hatch Survival at the End of Exposure

Treatment (mg/L)	Total Hatched	Survived	Dead	% Post-hatch survival
Pooled Control	154	144	10	94
0.0046	76	76	0	100
0.010	77	69	8	90
0.022	80	70	10	88
0.046	76	71	5	93
0.10	77	71	6	92

Table 4: Mean Body Weight (mg) and Percentage of Reduction at the End of Exposure

Treatment (mg/L)	Mean	Std. Dev.	n	%Reduction
Pooled Control	79.351	6.7572	8
0.0046	70.411	3.9003	4	11.3
0.010	72.843	4.2602	4	8.2
0.022	70.993	7.2041	4	10.5
0.046	74.561	10.1240	4	6.0
0.10	70.842	5.7372	4	10.7

 

Table 5: Mean Body length (mm) and Percentage of Reduction at the End of Exposure  

Treatment (mg/L)	Mean	Std. Dev.	n	%Reduction
Pooled Control	21.09	0.485	8
0.0046	20.22	0.419	4	4.1*
0.010	20.63	0.419	4	2.2
0.022	20.62	0.538	4	2.2
0.046	21.13	0.954	4	-0.2
0.10	20.60	0.432	4	2.3

*Effect was statistically significant (p≤0.05)

Acceptability of the test

- The dissolved oxygen concentration was maintained above 60% of the air saturation value throughout the test;

- The average temperature measured at weekly intervals in the controls and the treatment groups was within the range described in the study plan: 25±1.5°C. The temperature continuously measured in one of the replicates of the control was generally within the range described in the study plan: 25±1.5°C, with four exceptionswhere minimum temperatures of 23.2, 23.3 and twice 23.4°C were measured.

- The overall survival of embryos at the end of hatching was 96% in the pooled control, which complies with the requirements of the guideline (i.e. at least 70% of the embryos in the control should hatch).

- The mean post-hatch larval survival was 94% in the pooled control. The validity criterion for post-hatch survival of at least 75% was met.

List of deviations

Sampling for Measurements of Test Concentrations; Analyses

-  The analytical method used for analysis of the samples taken during the study was based on project 20179377 for the development and validation of the analytical method.
Evaluation: Inadvertently an old project number was stated in the study plan. Samples taken during the study were analysed conform the correct project.

Sampling for Measurements of Test Concentrations; Number of Samples

- Range-finding test: No samples were taken from the residue left after filtration of the stock solutions.
Evaluation: Concentrations could be measured in the test solutions. Therefore, analysis of the residue was not necessary.

Test Procedure and Conditions; Temperature

-  Early life stage test: Temporary deviations from the maximum level of temperature occurred in the control vessel (continuous measurement), i.e. at Day 1, Day 3, Day 5 and Day 17. The minimum temperature reached was 23.2°C. Time periods were temperature was between 23.2 and 23.5°C ranged between 1 and 4.15 hours.
Evaluation: These were only slight deviations of the optimum range and for relatively short periods. Temperature changes were gradual and did not affect the toxicity of the test item on the fish.

Validity criteria fulfilled:

yes

Conclusions:

1,3-Bis (4-hydroxy benzoyl) benzene did not affect embryonic or larval survival and growth of Pimephales promelas at the maximum solubility in test medium, i.e. at 0.10 mg/L, after 32 days of exposure under flow-through conditions (NOEC).

Executive summary:

The chronic toxicity of 1,3-Bis (4-hydroxy benzoyl) benzene to the freshwater fish species Pimephales promelas was investigated in a GLP-compliant study performed in accordance with OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test). In addition, due to the low solubility of the test item, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The test was performed using a flow-through system with nominal concentrations of 0.0046, 0.010, 0.022, 0.046 and 0.10 mg/L, which were based on the results of a preceding range-finding test. A blank- and a solvent control were also included.

The test was performed with four replicates containing 20 embryos per replicate for each concentration and the control groups. During the embryonic and larval phases, the embryos/larvae were observed for survival and effects on development, appearance and swimming behaviour. At the end of the test, the surviving fish were measured and weighed. The test duration was 32 days.

Samples for chemical analysis of the actual 1,3-Bis (4-hydroxy benzoyl) benzene concentrations were taken at regular intervals during the test and showed that measured concentrations were in agreement with or slightly above nominal (91- 118%). The effect parameters could therefore be based on the nominal concentrations

Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations. The results led to the following conclusions for the substance:

- the hatching success (embryonic survival) was not affected at concentrations up to and including 0.10 mg/L (NOEC). Hence the LOEC was >0.10 mg/L.

- Post-hatch survival (larval survival) was not affected at concentrations up to and including 0.10 mg/L (NOEC). Hence the LOEC was >0.10 mg/L.

- Growth of the epxosed larvae was not affected at concentrations up to and including 0.10 mg/L (NOEC). Hence the LOEC was >0.10 mg/L for both body weight and body length.

The reported effect concentrations are based on nominal concentrations.

Description of key information

Since no toxicity study in fish is available for 1,4 -Bis(4 -hydroxy benzoyl) benzene, the results from the structural analogue 1,3-Bis(4 -hydroxy benzoyl) benzene are used instead (for details see Read-across justification as attached in section 13).

The chronic toxicity to the freshwater fish species Pimephales promelas of the substance 1,3 -Bis (4 -hydroxy benzoyl) benzene was investigated under flow-through conditions in a GLP-compliant study performed in accordance with OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test). In addition, due to the low solubility of the test item, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations. The results led to the following conclusions for the substance:

- the hatching success (embryonic survival) was not affected at concentrations up to and including 0.10 mg/L (NOEC). Hence the LOEC was >0.10 mg/L.

- Post-hatch survival (larval survival) was not affected at concentrations up to and including 0.10 mg/L (NOEC). Hence the LOEC was >0.10 mg/L.

- Growth of the epxosed larvae was not affected at concentrations up to and including 0.10 mg/L (NOEC). Hence the LOEC was >0.10 mg/L for both body weight and body length.

The reported effect concentrations are based on nominal concentrations.

Additional information

The chronic toxicity of 1,3 -Bis (4 -hydroxy benzoyl) benzene to the freshwater fish species Pimephales promelas was investigated under flow-through conditions in a GLP-compliant study performed in accordance with OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test). The study is considered as reliable (Klimisch 1) and was selected as key study for the endpoint.

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