Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January - 17 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
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
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:
- Sampling: 1.0 mL samples for analysis were taken in duplicate from all test concentrations and the control at the beginning and at the end of two intervals of 48 hours (nominal days 0 and 2, 19 and 21) and one interval of 72 hours (nominal days 9 and 12).
- 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:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a loading rate of 1.0 mg/L applying a two-day period of magnetic stirring to reach the maximum dissolution of the test item in medium. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter (RC55, Whatman) where after the obtained saturated solution (SS) was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives
- Evidence of undissolved material: no, all final test solutions were clear and colourless
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: In-house laboratory culture with a known history
- Age of parental stock: young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Feeding during test: Food type, frequency and amount: Twice daily an amount of 0.25 mL of a Chlorella sp.suspension. On weekend days an amount of 0.50 mL was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day.

Breeding:
Start of each batch is with newborn daphnids, i.e. less than 3 days old, by placing by placing them individually in 50 ml M7-medium.
Maximum age of the cultures: 4 weeks.
Renewal of the cultures: Three times a week the young are counted and the parental daphnids are transferred to new media.
Feeding: daily, a suspension of fresh water algae
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
180 mg/L as CaCO3
Test temperature:
19-20°C (test vessels)
18-20°C (control vessel)
pH:
7.6 - 8.3
Dissolved oxygen:
7.5 - 9.8 mg/L
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100% of a saturated solution prepared at 1.0 mg/L.
Measured: 0.019, 0.033, 0.059, 0.091 and 0.16 mg/L calculated as time weighted mean.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 ml (6 x Ø 3.5 cm) volume, all-glass covered with a Perspex plate.
- Aeration: not indicated
- Renewal rate of test solution: three times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Test medium: M7
- Source/preparation of dilution water: tap water purified by Reverse Osmosis
- Total organic carbon: n.d. (measured at the start of the test, using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu ASI-V auto sampler.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily
- Light intensity: at the start: 615-630 lux; at the end: 571-677 lux

EFFECT PARAMETERS MEASURED:
Parental daphnids
Condition: Every workday the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday
Body length: At the end of the test.
Offspring
Appearance first brood: When observed.
Newborn daphnids: Every workday the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: A 10-day range-finding test was included. The concentrations of 1,3-Bis (4-hydroxy benzoyl) benzene were: 1.0, 10 and 100% of a SS at 1.0 mg/L. A control group was also included. Each concentration consisted of two replicates containing a total of ten daphnids (five each). The total test period was ten days. Test conditions were held as similar as possible to those applied in the reproduction test including feeding. Test solutions were renewed on days 1, 4, 6 and 8 during the test. Samples for possible analysis were taken at days 0 (fresh), 1 (fresh and old), 4 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions, but without daphnia and algae. Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions without daphnia and algae. No parental mortality, immobile young or aborted eggs were observed at any of the test concentrations. In the control, one parental daphnid was found dead after two days of exposure. No immobile young or aborted eggs were found in the control during the 10-day exposure period. At the end of the renewal periods all concentrations were in agreement with the initial concentrations or slightly higher, i.e. 95-128% of initial after 1 day, 113-124% of initial after 2 days and 115- 149% of initial after 3 days. The presence of organisms did not have an effect on the actual dissolved concentrations in test medium, i.e. concentrations in solutions with and without organisms were similar.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
not required
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.16 ng/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Measured concentrations: The analytical support confirmed that the freshly prepared test concentrations at days 0, 9 and 19 were in the range of 66-97% compared to each other. These concentrations remained stable during the renewal periods, i.e. 81-105% of initial, except for the concentration in 56% of the SS that was 71% of initial at the end of the last renewal period. Given these results the time weighted mean (TWM) concentrations were calculated and used for the determination of the effect parameters. Small responses at the retention time of the test item were detected in the chromatograms of the blank QC samples. They were considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks. The maximum contribution to the 0.01 mg/L QC samples was 1.7%.
- Parental mortality (see Table 1 of any other information on results): Three out of the twenty parental daphnids exposed to the control died during the test period. Hence, parental mortality did not exceed 20% in the control. 50% of the parental daphnids exposed to 0.091 mg/L died during the test period, while no mortality was observed in the higher concentration of 0.16 mg/L. Therefore, mortality at 0.091 mg/L was seen as inadvertent and not test item related. Statistical analysis showed that mortality was not different from the control group at any of the test concentrations.
- Age at first reproduction: No delay in the appearance of the first brood was observed in any of the concentrations tested when compared to the pooled control.
- Reproduction (see Table 2 of any other information on results): On average, 153 young were produced per survived daphnid in the control. Statistical analysis showed that the reproduction of the daphnids was not significantly different from the control group at any of the test concentrations.
- Body length (see Table 3 of any other information on results): No statistically significant reduction of growth was found at any of the test concentrations.
Reported statistics and error estimates:
Mortality of parental daphnids was analysed with Fisher`s exact binomial test with Bonferroni correction (one-sided greater, alpha = 0.05) and qualitative trend analysis by contrasts (monotonicity of concentration/response).
The following statistical procedures were used for reproduction and growth:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
- Differences between treatments and the control: Dunnett`s Multiple t-test procedure (onesided smaller, alpha = 0.05) after trend analysis by contrasts (monotonicity of concentration/response)
All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Mortality (Immobility) of parental daphnids at the end of the test

TWM concentration (mg/L)

Total introduced

Mobile

Immobile

% Immobility

0

20

17

3

15.0

0.19

10

9

1

10.0

0.033

10

0

0

0.0

0.059

10

10

0

0.0

0.091

10

5

5

50.0

0.16

10

10

0

0.0

Table 2: % Reduction of cumulative offspring per survived parent at Day 21

TWM concentration (mg/L)

Number of live females

Mean number of living young

Std. Dev.

Inhibition of reproduction (%)

0

17

152.8

13.06

 

0.19

9

154.9

12.60

-1.4

0.033

0

159.1

15.18

-4.1

0.059

10

154.8

19.66

-1.3

0.091

5

158.0

19.65

-3.4

0.16

10

160.3

14.75

-4.69

Table 3: Group mean body length (mm) and % reduction of length at Day 21

TWM concentration (mg/L)

Number of live females

Mean body lenth (mm)

Std. Dev.

% Reduction of Length at Day 21

0

17

4.37

0.201

 

0.19

9

4.41

0.118

-0.8

0.033

0

4.39

0.145

-0.3

0.059

10

4.33

0.131

0.9

0.091

5

4.32

0.185

1.2

0.16

10

4.33

0.101

1.0

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Remarks:
see "overall remarks"
Conclusions:
1,3-Bis (4-hydroxy benzoyl) benzene did not affect reproduction, parental mortality and growth of Daphnia magna at the maximum solubility in test medium, i.e. at 0.16 mg/L, after 21 days of exposure (NOEC).
Executive summary:

The long-term toxicity of the test item to aquatic invertebrates was studied in a Daphnia magna reproduction study according to OECD 211 and under GLP conditions The test was performed in a semi-static system, including 10 vessels per test concentration and 20 vessels for the control group. Each of the vessels contained one neonate (<24 h old) in 50 mL test medium. The nominal test concentrations were 10, 18, 32, 56 and 100% of a saturated solution (SS) prepared at 1.0 mg/L. The study duration was 21 days and the test solutions were renewed three times a week. Parental mortality, number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Samples taken at the beginning and the end of two intervals of 48 hours and one interval of 72 hours were analyzed. The analytical results confirmed that the freshly prepared test concentrations at days 0, 9 and 19 were in the range of 66-97% compared to each other. These concentrations remained stable during the renewal periods, i.e. 81-105% of initial, except for the concentration in 56% of the SS that was 71% of initial at the end of the last renewal period. Given these results the time weighted mean (TWM) concentrations were calculated to be 0.019, 0.033, 0.059, 0.091 and 0.16 mg/L.

Under the conditions of the present study 1,3-Bis (4-hydroxy benzoyl) benzene did not affect reproduction, parental mortality and growth of Daphnia magna at the maximum solubility in test medium, i.e. at 0.16 mg/L, after 21 days of exposure (NOEC). The study met the validity criteria and is considered reliable without restrictions.

Description of key information

The long-toxicity of the 1,3-Bis (4-hydroxy benzoyl) benzene to the freshwater flea Daphnia magna was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 211. 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. Under the conditions of the present study 1,3-Bis (4-hydroxy benzoyl) benzene did not affect reproduction, parental mortality and growth of Daphnia magna at the maximum solubility in test medium, i.e. at 0.16 mg/L, after 21 days of exposure (NOEC). The study met the validity criteria and is considered reliable without restrictions.

Key value for chemical safety assessment

Additional information

The long-term toxicity of the test item to aquatic invertebrates was studied in a Daphnia magna reproduction study according to OECD 211 and under GLP conditions. The study is considered as reliable (Klimisch 1) and is selected as the key study for this endpoint.