6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

Long-term toxicity to aquatic invertebrates

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:

03 April 2017 to 24 April 2017

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:

OECD Guidelines for Testing of Chemicals, No.: 211 (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Concentration of LOWINOX® 44B25 in the test solution was determined - when freshly prepared and at renewal - on one occasion during the first week. These determinations were repeated at weekly intervals thereafter.
The concentration of the test item was determined by HPLC-UV method.

Duplicate samples were taken (2-2 mL) in plastic tubes at the beginning and at the end of the experiment from three replicates at the applied test concentration level as well as from the control at the following time points:
1. week: on day 0 (fresh media, start of the experiment) and on day 1 (old media)
2. week: on day 9 (fresh media) and on day 10 (old media)
3. week: on day 20 (fresh media) and on day 21 (old media, end of the experiment)

After sampling, samples were frozen and kept approximately at -20°C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set is retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).
Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.0088 mg/L) at each measurement during the experiment, consequently biological results were based on the nominal concentrations.

Vehicle:

no

Details on test solutions:

Because the test item is poorly soluble in water, a test solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solution (100 mg/L nominal loading rate WAF) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days before the start of the test. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C.
The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give appropriate 100 mg/L nominal loading rate WAF.
Since the test was performed under semi-static conditions, prior to the treatment, at each of the renewal periods, test item solution was prepared by the method described above.
Test solutions renewals were performed at 24-hour intervals during the experiment.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna Straus
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Stock culture: Cultures was used for the test do not contain ephippia.
Breeding Conditions: Daphnia magna are bred in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of strain: The Daphnia magna is the standard species of reproduction test to be used according to the OECD guideline 211.
Number of animals: Ten animals were tested in every concentration and in the control as well during the main experiment.
Loading: Parent animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.
Age of animals: Less than 24 hours old at the beginning of the test and not first brood progeny.
Acclimatisation: To avoid the necessity of adaptation prior to the test the quality of water used was similar to the culture media.
Parent animals: The animals present at the start of the test and their reproductive output was the object of this study.
Offspring: Produced by the parental animals in the course of this test. The parental animals were 8-10 days old during the test when the first young Daphnids emerged from the brood pouch, after which a new batch appears every two to three days generally. These batches are called "broods" of the F1 (filial 1) generation.
Water: For culturing Daphnia, Elendt M4 media was used.

Food and Feeding
The Daphnia were fed daily with concentrated algal suspension of Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The supplied diet was based on the amount of organic carbon (C) provided to each parent animal. The constant ration level was 0.15 mg C/Daphnia/day during the test.
The cell number of algal-suspension fed was counted before the test. The total organic carbon content (TOC) of algal-suspension fed was based on the results of the last annual nomographical TOC measurement.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

No post exposure period specified in the study report.

Hardness:

Hardness was above 140 mg/L (as CaCO3).

Test temperature:

The temperature of the test media and in the climate chamber was within the range 18 - 22°C.

pH:

The pH was within the range 6 - 9.

Dissolved oxygen:

The dissolved oxygen concentration was above 3 mg/L at the beginning and during the test.

Salinity:

Not applicable.

Conductivity:

Not specified.

Nominal and measured concentrations:

Nominal concentrations.

Details on test conditions:

Untreated Control
The dilution water (Elendt M4) was used as a test solution without addition of the test item.

Test Water
Elendt M4 (according to OECD 211) was used as dilution water for both the range finding and definitive tests. The same composition of dilution water was used for the tests and for breeding the test animals.

ADMINISTRATION OF THE TEST ITEM
Concentrations
A 10-days preliminary range finding test was conducted to determine the approximate toxicity of the test item on the reproductive output of Daphnia, so that appropriate test concentrations can be selected for use in the definitive test. 5 test animals were exposed in every concentration level and also in the control.

Justification of the Concentrations
Because significant effect was not observed at the highest examined concentration level during the 10-days preliminary range-finding test, a limit test was carried out using 100 mg/L loading rate WAF and one control were investigated in the definitive test.

PERFORMANCE OF THE TEST
This 21-days reproduction test was carried out in a semi-static renewal system, with a renewal frequency of 24 hours. The frequency of test solutions renewal was based on the stability of the test substance and performed preliminary test.
When the medium was renewed a second series of test vessels were prepared and the parent animals transferred to them by a pipette of suitable diameter. The volume of test medium transferred with the Daphnia was minimised.

Observations
Mortality of the parent animals was recorded daily during the test. The living offspring produced by each parent animal were removed and counted daily from the appearance of the first brood.
Measurement of the length of the parent animals (excluding the anal spine) was performed at the end of the test.

Test conditions
A light-dark cycle was necessary: 8 hours darkness and 16 hours light. The light intensity was within the range of 15-20 μE m-2s-1 and it was checked at the start of the test.
Temperature, oxygen concentration and pH values were measured at the start of the experiment, thereafter at each renewal period in fresh and old media, in the control and in the test item concentration in one replicate.
Hardness was determined in fresh and old media, in the control as well as in the test item concentration at each renewal period. The temperature of the test media and in the climate chamber was within the range 18 – 22 °C, and the deviation from the mean temperature value was less than ± 2 °C. The dissolved oxygen concentration was above 3 mg/L at the beginning and during the test. The pH was within the range 6 - 9, it did not vary by more than 1.5 units during the study period.
Test vessels were not aerated during the test.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 other: mg/L nominal loading rate

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg /L nominal loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

VALIDITY
There was no mortality in the control group. The mean number of live offspring per parent animal surviving in the control at the end of the test was 144.20 with a variation coefficient of 20.21 %. No ephippia was observed during the test.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

TEST CONDITIONS
Light period: 16 hours photoperiod / 8 hours dark, daily
Light intensity: 1283.9 lux (17.35 μE m-2s-1)
Temperature:
19.5 – 20.6 °C (in the climate chamber)
20.1 –20.5 °C (in the fresh media)
19.9 –20.3 °C (in the old media)
pH values:
7.42 – 7.99 (in the fresh media)
7.64 – 8.29 (in the old media)
Oxygen concentration:
7.5 – 8.7 mg/L (in the fresh media)
8.4 – 9.7 mg/L (in the old media)
Hardness:
243 – 277 mg/L (in the fresh media)
250 – 279 mg/L (in the old media)

MORTALITY OF PARENTS
Mortality was not observed neither in the control nor in the examined test item treated group (100.0 mg/L WAF) during the experiment.

JUVENILE PRODUCTION
The mean number of live offspring per parent animal surviving in the control at the end of the test was 144.20 with a variation coefficient of 14.01 %.
Statistically significant difference was observed concerning the time to production of first brood between the examined test item treated group (100.0 mg/L WAF) and the control group. However, taking into account that this deviation was in positive direction, it cannot be considered to be related to treatment with test item.
The reproduction of the surviving parent animals was not significantly different from the control group in the examined test item treated group of 100.0 mg/L loading rate WAF (2 Sample t-Test, α = 0.05).
There was a statistically significant differences between the reproductive output of the parent animals per day at the test item treated group when compared to the control (2 Sample t-Test, α =0.05).
Aborted eggs, dead offspring, males or ephippia were not found during the study.

BODY LENGTHS OF PARENT ANIMALS
The measurement of the length of the surviving parent animals was performed at the end of the test (Day 21).
The body length of the surviving parent animals in the test item treated group was significantly different from the control group (2 Sample t-Test, α =0.05).

Results with reference substance (positive control):

Positive control not used for this study.

Reported statistics and error estimates:

To decide if there was an effect of the test item on reproductive output the mean value and standard deviation of the reproductive output across replicates of the test concentration were calculated.
For the estimation of the LOEC, and hence the NOEC, for effects of the test item on reproductive output the mean value and standard deviation of the reproductive output across replicates of test concentration were calculated using analysis of variance (ANOVA). The mortality, the mean body length of test animals and the mean reproduction at test item concentration was compared with the control mean using an appropriate comparison method (2 Sample t-Test, α = 0.05).
As a limit test was performed the EC50 value (and EC20 or EC10 as well) was not calculated and determined from the raw data.

Statistically significant difference was also observed concerning the time to production of first brood between the test item group of 100.0 mg/L WAF and the control group. However, taking into account that this deviation was in positive direction, it cannot be considered to be related to treatment with test item.

Results of the Preliminary Range-Finding Test

Nominal loading rate WAFs [mg/L]	Untreated control	1	10	100
Number of treated / immobilised animals	5 / 0	5 / 0	5 / 0	5 / 0
Number of living newborns [mean]	10.0	11.0	13.4	9.2
Reduction (%)	-	-10.0	-34.0	8.0

 

Mortality of parent animals (Daphnia magna)

Parameter (Day 21)	Results (mg/L loading rate WAF, nominal)
LC10	>100.0
LC20	>100.0
LC50	>100.0
NOEC (Mortality)	100.0
LOEC (Mortality)	>100.0

 

Juvenile reproduction of parent animals (Daphnia magna) (number of living newborns during the test)

Parameter (Day 21)	Results (mg/L loading rate WAF, nominal)
Reproduction EC10 95% conf. limits	>100.0 (could not be determined)*
Reproduction EC20 95% conf. limits	>100.0 (could not be determined)*
Reproduction EC50 95% conf. limits	>100.0 (could not be determined)*
NOEC (Reproduction) LOEC (Reproduction)	100.0 >100.0

*Remark: As a limit test was performed the EC50 value (and EC20 or EC10 as well) was not calculated and determined from the raw data.

 

Summary of reproductive output of the parent animals

Test group	Mean number of living offspring per surviving parent animal	SD	CV (%)	Reduction (%)	Significance
Control	144.20	20.21	14.01	-	-
100.0 mg/L WAF (nominal)	130.30	7.72	5.92	9.6	n.s.

SD: standard deviation

CV (%): variation coefficient

n.s.: statistically not significant compared to the control (2 sample t-Test, α=0.05)

 

Summary of reproductive output of the parent animals per day

Test group	Mean number of living offspring per surviving parent animal per day	SD	CV (%)	Reduction (%)	Significance
Control	10.85	1.45	13.34	-	-
100.0 mg/L WAF (nominal)	9.31	0.55	5.92	14.2	*

SD: standard deviation

CV (%): variation coefficient

*: statistically significantly different compared to the control (2 sample t-Test, α=0.05)

 

Mean body length of the survival parent animals at the end of the test

Test group	Mean body length of parents surviving at the end of the test (mm)	SD	CV (%)	Reduction (%)	Significance
Control	4.74	0.34	7.25	-	-
100.0 mg/L WAF (nominal)	4.17	0.32	7.75	12.0	*

SD: standard deviation

CV (%): variation coefficient

*: statistically significantly different compared to the control (2 sample t-Test, α=0.05)

Validity criteria fulfilled:

yes

Conclusions:

In this 21-days Reproduction test on Daphnia magna with LOWINOX® 44B25 the 21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 values were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.

Executive summary:

The purpose of this study was to estimate the effect of the test item LOWINOX® 44B25 on the reproductive output of Daphnia magna determining the NOEC, LOEC, EC50, EC20 and EC10 for reproduction after 21 days of exposure.

Daphnia magna were exposed in a semi-static test to saturated aqueous test media containing the test item at 100.0 mg/L nominal loading rate WAF in a limit test for 21 days.

The test method of application and the test species Daphnia magna are recommended by the test guidelines.

 

This 21-days reproduction test was carried out in a semi-static renewal system, with a renewal frequency of 24 hours based on analytical method validation (Study Code: 16/446-901AN) (due to that test item is a UVCB substance with very poor solubility).

 

Concentration of the test item in the test solution was analysed by the validated method - when freshly prepared and at renewal - on one occasion during the first week then these determinations were repeated weekly intervals thereafter based on the performed analytical method validation. Fully defined Elendt M4 medium was used as dilution water for the test solution.

Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.0088 mg/L) for all WAF samples (freshly prepared and at renewal) during the experiment. The biological results are based on the nominal loading rate WAF.

 

Test item exposure and control group comprised 10 Daphnids. The test animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.

 

The time to the production of first brood and the numbers of broods per animal were recorded during the test. The measurement of the length of the surviving parent animals was performed at the end of the test.

 

The average cumulative number of young per parent animal in the control was 144.20 with a variation coefficient of 14.01 %.

 

Mortality was not observed neither in the control or in the test item treated group of 100.0 mg/L nominal loading rate WAF during the whole experiment.

 

The reproduction of the surviving parent animals was not significantly different from the control group in the test item treated group of 100.0 mg/L nominal loading rate WAF (2 Sample t-Test, α = 0.05).

The body length of the surviving parent animals was significantly different from the control group in the test item treated group of 100.0 mg/L nominal loading rate WAF (2 Sample t-Test, α = 0.05).

Statistically significant difference was also observed concerning the time to production of first brood between the test item group of 100.0 mg/L WAF and the control group. However, taking into account that this deviation was in positive direction, it cannot be considered to be related to treatment with test item.

 

In this 21-days Reproduction test on Daphnia magna with LOWINOX® 44B25 the 21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 values were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.

Description of key information

21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 values were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

This 21-days reproduction test was carried out in a semi-static renewal system, with a renewal frequency of 24 hours based on analytical method validation (Study Code: 16/446-901AN) (due to that test item is a UVCB substance with very poor solubility). 

Concentration of the test item in the test solution was analysed by the validated method - when freshly prepared and at renewal - on one occasion during the first week then these determinations were repeated weekly intervals thereafter based on the performed analytical method validation. Fully defined Elendt M4 medium was used as dilution water for the test solution.

Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.0088 mg/L) for all WAF samples (freshly prepared and at renewal) during the experiment. The biological results are based on the nominal loading rate WAF.

 

Test item exposure and control group comprised 10 Daphnids. The test animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.

The time to the production of first brood and the numbers of broods per animal were recorded during the test. The measurement of the length of the surviving parent animals was performed at the end of the test.

 

The average cumulative number of young per parent animal in the control was 144.20 with a variation coefficient of 14.01 %.

Mortality was not observed neither in the control or in the test item treated group of 100.0 mg/L nominal loading rate WAF during the whole experiment.

 

The reproduction of the surviving parent animals was not significantly different from the control group in the test item treated group of 100.0 mg/L nominal loading rate WAF (2 Sample t-Test, α = 0.05).

The body length of the surviving parent animals was significantly different from the control group in the test item treated group of 100.0 mg/L nominal loading rate WAF (2 Sample t-Test, α = 0.05).

Statistically significant difference was also observed concerning the time to production of first brood between the test item group of 100.0 mg/L WAF and the control group. However, taking into account that this deviation was in positive direction, it cannot be considered to be related to treatment with test item.

 

In this 21-days Reproduction test on Daphnia magna with LOWINOX® 44B25 the 21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 values were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.