Butanedioic acid, 2-sulfo-, 1(or 4)-[2-(C12-18 and C18-unsatd. amido)ethyl] esters, disodium salts

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from each treatment and control group at test initiation, at the beginning and the end of the longest renewal cycle each week, and at test termination. Samples of “new” solutions were collected from the bulk preparations of each test solution at test initiation and at the beginning of the longest renewal cycle. Samples of “old” solutions were collected from two alternating replicates of each treatment and control group at the end of the longest renewal cycle and at test termination. The samples (8.00 mL) were collected from mid-depth, placed in glass vials containing 2.00 mL of 0.5% formic acid in methanol, and processed immediately for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Test solutions were prepared every 2 to 3 days (typically each Monday, Wednesday and Friday) during the test. All test solutions were adjusted to 100% active ingredient during preparation, based on test item purity (39.7%). Prior to removing an aliquot for stock solution preparation, test item container was vortexed and inverted to ensure a homogeneous mixture. A primary stock solution was prepared in AAP media at a nominal concentration of 10 mg a.i./L, equivalent to the highest concentration tested, by sonicating a calculated amount of test substance in 3000 mL of AAP media for approximately 35 minutes followed by inversion for at least 20 times. After preparation the primary stock solution appeared clear and colorless, with bubbles on the surface of the solution. Proportional dilutions of the primary stock solution were made in AAP media to prepare test solutions at nominal concentrations of 0.13, 0.42, 1.3, 3.8 and 12 mg a.i./L. The test solutions were mixed by inversion, and approximately 200-mL aliquots were added to each test chamber. All test solutions appeared clear and colorless, while bubbles were noted on the surface of the 3.8 and 12 mg a.i./L treatment concentrations. No visible precipitate was noted in any of the test solutions.

- Controls: blank medium (AAP medium)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Feeding during testDuring culturing and testing, daphnids were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcapitata. At each feeding, each test chamber was fed 0.50 mL of YCT, 1.0 mL of algae and 0.40 mL of vitamin solution. This amount of feed is equal to approximately 0.6 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the system to support acceptable reproduction rates.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The 3 adult daphnids used to supply neonates for the test were held for at least 20 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7 day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. To initiate the test, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and individual neonates were transferred to each test chamber to initiate the test. All transfers were made below the water surface using wide-bore pipettes.

Test type:

semi-static

Water media type:

freshwater

Remarks:

AAP medium was used because this substance class has better solubility in this medium when compared to the typical Daphnia medium (Elendt M4 or M7 or ISO)

Limit test:

no

Total exposure duration:

21 d

Hardness:

4 to 20 mg/L as CaCO3

Test temperature:

Temperature monitored continuously in a container of water placed adjacent to the test chambers ranged from 19.36 to 20.41ºC.

pH:

7.0 - 8.9

Dissolved oxygen:

6.9 to 9.1 mg/L (A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20ºC in freshwater.)

Conductivity:

94 - 114 µS/cm

Nominal and measured concentrations:

Nominal Concentration based on Active Ingredient: Negative Control; 0.13, 0.42, 1.3, 3.8, 12 mg a.i./L
Time-Weighted Mean Measured Concentration of C12 Component in Test Item: < LOQ, 0.092, 0.18, 0.42, 1.4, 5.1 mg a.i./L
Time-Weighted Mean Measured Concentration of C14 Component in Test Item: < LOQ, 0.091, 0.18, 0.40, 0.97, 3.6 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test chambers were 250-mL glass beakers loosely covered with plastic petri dishes, and contained approximately 200 mL of test solution. The depth of solution in a representative test chamber was 6.5 cm. The test chambers were impartially positioned in an environmental chamber to maintain the target water temperature throughout the test period. All test chambers were labeled with the project number, test concentration and replicate designation

- Aeration: no
- Renewal rate of test solution (frequency): Test solutions were prepared every 2 to 3 days (typically each Monday, Wednesday and Friday) during the test.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
AAP medium was used because this substance class has better solubility in this medium when compared to the typical Daphnia medium (Elendt M4 or M7 or ISO).
The daphnids were cultured and tested in freshwater AAP medium based on the sponsor’s request. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified well water (NANOpure® water). The test medium was then prepared by adding appropriate volumes of the stock nutrient solutions to NANOpure® water (4). The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid (HCl) as needed.
- Culture medium different from test medium: no
- Intervals of water quality measurement: once a week

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod and Light intensity:
Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
survival (mortality), reproduction, growth


TEST CONCENTRATIONS
- Spacing factor for test concentrations: about 3
- Justification for using less concentrations than requested by guideline: NA
- Range finding study: 13 day range finder
- Test concentrations: control, 0.23, 0.76, 2.5, 8.4, 28 mg a.i./L
- Results used to determine the conditions for the definitive study:
survival 100, 80, 100, 100, 0, 60 percent
reproduction: 82.4, 72.5, 79., 88.9, 0, and 51.3 Mean Number of Young Produced Per Reproductive Day

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C12 constituent

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C12 constituent

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

C12

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C12 constituent

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C14 constituent

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C14 constituent

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C14 constituent

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on measurement of C14 constituent

Basis for effect:

growth

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: see table below
- Other biological observations: none
- Mortality of control: 10%
- Other adverse effects control: no
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

See "Any other information on materials and methods" (above).

The cladoceran,Daphnia magna,was exposed to the test substance, the aqueous solution of butanedioicacid, 2 (or 3)-sulfo-, 4-[2-[(1-oxo (C12-C18 (Even Numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts,at time-weighted mean measured concentrations of 0.092, 0.18, 0.42, 1.4 and 5.1 mg a.i/L based on C12 component and from 0.091, 0.18, 0.40, 0.97 and 3.6 mg a.i./L based on C14 component under semi-static conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤5.1 mg a.i./L based on C12 component or ≤3.6 mg a.i./L based on C14 component of the test item. Consequently, the NOEC, LOEC and EC₁₀, EC₅₀and the corresponding 95% confidence interval were as follows:

 

	Survival	Reproduction (mean number of live neonates)		Length	Dry Weight
		Per Surviving Adult	Per Adult at Start
Based on time-weighted mean measured concentration of C12 component (mg a.i./L)^1
EC10& 95% confidence interval	--	>5.1 (NA)	>5.1 (NA)	--	--
EC50& 95% confidence interval	>5.1 (NA)	>5.1 (NA)	>5.1 (NA)	--	--
NOEC	5.1	5.1	5.1	5.1	5.1
LOEC	>5.1	>5.1	>5.1	>5.1	>5.1
Based on time-weighted mean measured concentration of C14 component (mg a.i./L)^1
EC10& 95% confidence interval	--	>3.6 (NA)	>3.6 (NA)	--	--
EC50& 95% confidence interval	>3.6 (NA)	>3.6 (NA)	>3.6 (NA)	--	--
NOEC	3.6	3.6	3.6	3.6	3.6
LOEC	>3.6	>3.6	>3.6	>3.6	>3.6

¹  Mean measured test concentrations were calculated using time-weight average as described in OECD 211, Annex 6.

   NA = Not applicable; the 95% confidence intervals were overly wide or outside of the data range used for the calculation.

   -- = not estimated as it was not required.

Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to the Test Item for 21 Days

 

Nominal Test Concentration (mg a.i./L)	Time-Weighted Mean Measured Concentration (mg a.i./L)		Percent Adult Survival^1	Mean No. Neonates Per Surviving Adult (# Surviving Adults) ± Std. Dev.^1,3	Mean No. Neonates Per Adult at Start (# Surviving Adults) ± Std. Dev.^2	Mean Length ± Std. Dev. (mm)^1	Mean Dry Weight ± Std. Dev. (mg)^1
	C12	C14
Negative Control	Negative Control	Negative Control	90	169 ± 25.1 (n=18) (CV = 14.9%)	155 ± 48.4	5.1 ± 0.14	1.06 ± 0.21
0.13	0.092	0.091	60	160 ± 22.0 (n=6)	145 ± 50.0	5.0 ± 0.16	1.03 ± 0.17
0.42	0.18	0.18	80	176 ± 14.4 (n=8)	152 ± 52.2	5.0 ± 0.14	0.95 ± 0.19
1.3	0.42	0.40	70	180 ± 28.4 (n=7)	137 ± 76.9	5.0 ± 0.22	0.97 ± 0.27
3.8	1.4	0.97	90	167 ± 17.7 (n=9)	163 ± 21.1	4.9 ± 0.17	1.09 ± 0.21
12	5.1	3.6	70	166 ± 31.9 (n=7)	128 ± 69.9	4.9 ± 0.16	1.06 ± 0.11
^1  There were no statistically significant differences from the control (Fisher Exact test, p> 0.05 for survival and Dunnett’s one-tailed test, p > 0.05 for mean number of neonates per surviving adult and growth). ^2  There were no statistically significant differences from the control (Jonckheere-Terpstra step down trend test, p > 0.05). ^3There were total of 15, 8, 6, 8, 6 and 7 immobile neonates produced and total of 14, 28, 11, 10, 6 and 9 aborted/shed eggs produced by surviving adults in the negative control, 0.13, 0.42, 1.3, 3.8 and 12 mg a.i./L nominal test concentrations, respectively. These were production of immobile neonates and aborted/shed eggs not considered to be treatment related effect.

Validity criteria fulfilled:

yes

Conclusions:

the overall NOEC and EC10 were > 3.6 mg a.i./L and 3.6 mg/L respectively.

Executive summary:

 In the Klimisch 1 study from Minderhout (2018) the cladoceran,Daphnia magna,was exposed to the aqueous solution of butanedioic acid, 2 (or 3)-sulfo-, 4-[2-[(1-oxo (C12-C18 (Even Numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts,at time-weighted mean measured concentrations of 0.092, 0.18, 0.42, 1.4 and 5.1 mg a.i/L based on C12 component and from 0.091, 0.18, 0.40, 0.97 and 3.6 mg a.i./L based on C14 component under semi-static conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤5.1 mg a.i./L based on C12 component or ≤3.6 mg a.i./L based on C14 component of the test item. 

As worst case, the concentrations based on the measured C14 constituent were used for the risk assessment., i.e., the overall NOEC and EC10 were > 3.6 mg a.i./L and 3.6 mg/L respectively.

The results of this study are considered relevant and reliable for the risk assessment.

Description of key information

The overall NOEC and EC10 were > 3.6 mg a.i./L and 3.6 mg/L respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.6 mg/L

Additional information

 In the Klimisch 1 study from Minderhout (2018) the cladoceran,Daphnia magna,was exposed to the aqueous solution of butanedioic acid, 2 (or 3)-sulfo-, 4-[2-[(1-oxo (C12-C18 (Even Numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts,at time-weighted mean measured concentrations of 0.092, 0.18, 0.42, 1.4 and 5.1 mg a.i/L based on C12 component and from 0.091, 0.18, 0.40, 0.97 and 3.6 mg a.i./L based on C14 component under semi-static conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤5.1 mg a.i./L based on C12 component or ≤3.6 mg a.i./L based on C14 component of the test item. 

As worst case, the concentrations based on the measured C14 constituent were used for the risk assessment., i.e., the overall NOEC and EC10 were > 3.6 mg a.i./L and 3.6 mg/L respectively.

The results of this study are considered relevant and reliable for the risk assessment.