Docusate sodium

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guidline compliant study according to most recent guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L. Based on the biological results, samples from the nominal concentrations of 4.4 to 22 mg/L and the Control were analyzed.

- Sampling method:
For determination of the test item concentrations, quadruplicate samples (two 8 mL and two 10 mL samples) were taken from the freshly prepared test media of all test concentrations and from the control at one treatment period of the first, second and last week of the test (Day 0, 7 and 16, respectively). The 8 mL samples were treated with 2 mL methanol prior to sampling and were stored as backup. The 10 mL samples were used primarily for analytical measurements.

To determine the maintenance of the test item concentrations in the test media, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19).

The samples from the highest nominal concentration of 22 mg/L were taken on Days 0 and 2 as all daphnids were dead at observation on Day 5 at this concentration level.

The following stability samples were taken in quadruplicate (two 8 mL and two 10 mL samples):

a) Samples taken from the actual test by combining the contents of all replicate test beakers after the end of the test medium renewal period.
b) Samples incubated during the renewal periods under the test conditions, however without food and daphnids.

Immediately after the sampling, 2 mL methanol was added to the 8 mL samples.



- Sample storage conditions before analysis:
All samples were stored deep-frozen (at about –20 °C) till date of analysis. According to pre-experiments to the storage stability, the test item is sufficiently stable in test media samples under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prior to the start of the test and prior to each test medium renewal, the test medium of the highest nominal test concentration of 22 mg/L was freshly prepared by completely dissolving 66 mg of the test item (dosed in the range of 65.9 to 66.4 mg) in 3000 mL of test water using ultrasonic treatment for 15 minutes and stirring for 60 minutes at room temperature in the dark. The test medium of the highest test concentration was diluted with adequate volumes of test water to prepare the lower concentrated test media.
- Controls: Dilution water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none, dose verification indicated correct dosage and stability of the test solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: clone 5
- Source: A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium identical to the medium used for the test.
- Age of parental stock (mean and range, SD): at least 14 days but not older than 4 weeks.
- Feeding during test: yes
- Food type: The test animals were fed daily with a food mixture containing a suspension of green algae of the species Desmodesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use.
- Amount: The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.
- Frequency: daily

ACCLIMATION
- Acclimation period: not needed
- Acclimation conditions (same as test or not): similar conditions as in test, Each stock animal was maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.
- Type and amount of food: see description of test conditions

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

The water temperature was maintained at 20 21 °C

pH:

8.4 to 8.9

Dissolved oxygen:

at least 7.7 mg/L

Nominal and measured concentrations:

Nominal: Control, 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L, the biological data were based on nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: The test was performed in 100 mL glass beakers containing 80 mL of test medium.
- Type (delete if not applicable): The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions
- Material, size, headspace, fill volume: fill volume 80 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): every 48 or 72 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 daphnia / 80 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (Elendt M7 medium)
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the beginning and end of each test medium renewal period, the pH and dissolved oxygen concentrations were measured in one replicate of each test concentration and of the control. At the same time, the water temperature was measured in one of the control replicates. Additionally, the room temperature was continuously monitored. The appearance of the test media was visually inspected and recorded at the beginning and end of each test medium renewal period.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30 minute transition period
- Light intensity: Light intensity during the light period was between approximately 390 and 560 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95%CI 5.2 – 11 mg/L

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI 6.5 - 10 mg/L

Details on results:

- Mortality of parent animals: yes, used for EC10 calculation. In the control and at the lower test concentrations up to and including 4.4 mg/L, the survival of the test animals at the end of the test was at least 90% or higher. At the concentrations of 8.8 and 11 mg/L, the survival was 90 and 70%, respectively. At the highest test concentration of 22 mg/L, all test animals were dead at observation on Day 5. Mortality up to 20% is regarded as natural and tolerated by the test guideline. Thus, the survival of Daphnia magna after 21 days was reduced at the test concentrations of 11 and 22 mg/L.
- No. of offspring produced per day per female: The mean reproduction rate of the daphnids in the control was 137 ± 12 living offspring per surviving adult.No statistically significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the test concentration of 8.8 mg/L. From the concentration of 11 mg/L on, the mean reproduction rate of the adult daphnids was statistically significantly reduced to a mean value of 107 living offspring (78% compared to the control; Williams t-test, one-sided smaller, alpha = 0.05 based on transformed values). At the highest concentration tested (22 mg/L), no offspring was produced due to the mortality of the adult daphnids (see above).
- Body length and weight of parent animals: The mean body length of the daphnids in the control was 4.13 ± 0.08 mm (mean ± standard deviation. At all test concentration up to and including the test concentration of 11 mg/L, the mean body length was not statistically significantly smaller compared to the control (Williams t-test, one-sided smaller, alpha = 0.05). At the highest concentration tested (22 mg/L), no body length could be measured due to the mortality of the adult daphnids (see above).
- Type and number of morphological abnormalities: no visible abnormalities were observed at the test animals during the test
- Type and number of behavioural abnormalities: no visible abnormalities were observed at the test animals during the test
- Time to first brood release: The first young offspring released from their parent animals were recorded in the control and at the test concentrations up to and including 8.8 mg/L at observation on Day 7. At the next higher test concentration of 11 mg/L a delay of the first brood was observed (first offspring observed on Day 8 of exposure). At the highest concentration tested (22 mg/L), no offspring was produced due to mortality of the adult daphnids. Thus, the time of the first brood was not affected by the test item up to and including the test concentration of 8.8 mg/L.
- Effect concentrations exceeding solubility of substance in test medium: none

Reported statistics and error estimates:

The mean reproduction rates and mean body lengths of the daphnids at the test concentrations were both compared to the control by multiple Williams t-tests, one-sided smaller, alpha = 0.05. Additionally, the EC10 for the inhibition of the reproduction rate and the immobility after 21 days was calculated by Probit Analysis using linear maximum likelihood regression.

After 21 days 0, 10, 0, 0, 0, 10, 30 and 100% mortality was observed in the control and at nominal test concentrations of 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L, respectively. The mortality at 0.55 and 8.8 mg/L was considered not test item related. The reproduction per parent daphina was 137.0 (10), 135.6 (9*), 139.0 (10), 140.4 (10), 133.8 (10), 141.4 (9*), 107.4 (10) and 0 (10) living offspring per parental animal (number of parental daphinds used to calculate the mean are given in brackets) in the control and 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L, respectively.

Summary of the results after 21 days of exposure of the test animals:

	Control	Butanedioic acid, sulfo-, 1,4-bis(2-ethylhexyl)ester, sodium salt (nominal concentration [mg/L])
		0.55	1.1	2.2	4.4	8.8	11	22
Mortality [%] after 21 days of exposure	0	10**	0	0	0	10**	30	100
Mean reproduction rate (living offspring per adult)	137.0	135.6	139.0	140.4	133.8	148.1	107.4*	0*
Mean reproduction rate in % of control	100.0	98.9	101.5	102.5	97.7	108.1	78.4	0
Mean body length of the adults [mm]	4.13	4.11	4.16	4.16	4.17	4.17	4.19	n.a.
Mean body length of the adults in % of control	100.0	99.5	100.6	100.6	100.8	100.8	101.4	n.a.

 *      statistically significantly lower than the control value, results of a Williams t-test, one-sided smaller, alpha= 0.05 based on transformed values ** not considered test item related

n.a:.not applicable since all adult test animals died until Day 5

The biological results can be summarized as follows (on the basis of nominal concentrations of the test item):

 

Parameter	Inhibition of reproduction rate	Immobility
	(21 days)	(21 days)
EC10  [mg/L]	9.8	9.0
95% confidence interval	6.5 – 10	5.2 – 11
NOEC [mg/L]	8.8	8.8
LOEC [mg/L]	11	11

 

Validity criteria fulfilled:

yes

Conclusions:

21-day EC10: 9.0 mg/L

Executive summary:

In the Klimisch 1 GLP study from Eckenstein (2014) the chronic toxicity of Butanedioic acid, sulfo-, 1,4-bis(2-ethylhexyl)ester, sodium salt on Daphnia magna was determined in an 21 day semi-static test according to OECD 211. The test was performed with concentrations of 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L and a blank control. Ten replicates with 1 Daphnia each were set up. After 21 days 0, 10, 0, 0, 0, 10, 30 and 100% mortality was observed in the control and at nominal test concentrations of 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L, respectively. The reproduction per parent daphina was 137.0 (10), 135.6 (9*), 139.0 (10), 140.4 (10), 133.8 (10), 141.4 (9*), 107.4 (10) and 0 (10) living offspring per parental animal (number of parental daphinds used to calculate the mean are given in brackets). The measured concentrations of the test item in the test media of the test concentrations were between 94 and 110% of the nominal values in the freshly prepared test solutions and between 86 and 111% in the aged test solutions at the end of the renewal interval. Therefore, the biological results were based on nominal concentrations. The EC10 for reproduction is 9.8 mg/L, the EC10 for mortality is 9.0 mg/L. Length was not affected. For the risk assessment, the more conservative EC10, i.e., the EC10 for mortality was used.

The result is considered relevant and reliable for the risk assessment.

Description of key information

21-day EC10: 9.0 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9 mg/L

Additional information

In the Klimisch 1 GLP study from Eckenstein (2014) the chronic toxicity of Butanedioic acid, sulfo-, 1,4-bis(2-ethylhexyl)ester, sodium salt on Daphnia magna was determined in an 21 day semi-static test according to OECD 211. The test was performed with concentrations of 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L and a blank control. Ten replicates with 1 Daphnia each were set up. After 21 days 0, 10, 0, 0, 0, 10, 30 and 100% mortality was observed in the control and at nominal test concentrations of 0.55, 1.1, 2.2, 4.4, 8.8, 11 and 22 mg/L, respectively. The reproduction per parent daphina was 137.0 (10), 135.6 (9*), 139.0 (10), 140.4 (10), 133.8 (10), 141.4 (9*), 107.4 (10) and 0 (10) living offspring per parental animal (number of parental daphinds used to calculate the mean are given in brackets). The measured concentrations of the test item in the test media of the test concentrations were between 94 and 110% of the nominal values in the freshly prepared test solutions and between 86 and 111% in the aged test solutions at the end of the renewal interval. Therefore, the biological results were based on nominal concentrations. The EC10 for reproduction is 9.8 mg/L, the EC10 for mortality is 9.0 mg/L. Length was not affected. For the risk assessment, the more conservative EC10, i.e., the EC10 for mortality was used.

The result is considered relevant and reliable for the risk assessment.