Fatty acids, C12-14, .alpha.-sulfo, 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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

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: Ra-He 2014-054
- Expiration date of the lot/batch: 2018/10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed and dry; store in a cool place
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: 63.35g/L in water

Type of the test substance: No Unique and identified molecular structure substance, 100% UVCB
Chemical name : Fatty acids, C12-14, reaction product with sulfur trioxide, sodium salt
CAS: 2215087-54-8

Analytical monitoring:

yes

Details on sampling:

Oxygen concentration, temperature, hardness and pH values were measured once a week, both in fresh and old medium, in the blank control and in the treatment group.

During the test, when the medium was renewed in the semi-static test, a second series of test vessels had been prepared and the parent animals were transferred to them by a glass pipette of suitable diameter.
During the test, the measured concentrations of the test substance in the blank control and the treatment group were analyzed in samples taken from freshly prepared solutions and old solutions prior to renewal. The determination prior to renewal was only performed on one replicate vessel of each group. One test vessel was selected from each test vessel (i.e. replicate) randomly. Two 2mL test samples were taken respectively from the freshly prepared test solutions and the solutions prior to renewal at each group and filtrated with 0.22μm cellulose nitrate membrane filters. One was used to analyze and the other was stored as a back up.

Vehicle:

no

Details on test solutions:

10 Materials and reagents
10.1 Elendt M4 medium
Elendt M4 medium was used as test medium for the daphnia reproduction test. For the preparation of Elendt M4 medium stock solutions of the analytical grade reagents and deionized water were mixed in adequate volume ratios to get the final composition described in section 19.1. The Elendt M4 medium was stored at 2°C to 8°C for a maximum of 90 days. The concentration of TOC in the medium (i.e. before addition of the algae) was measured regularly every year and it was (M4) 1.19mg/L (target value below 2mg/L). The test medium was aerated for more than 30 minutes prior to the start of the test and placed at test temperature at least 12 hours before the test. For the medium at the beginning and during the range-finding test, the dissolved oxygen concentration was 8.11mg/L-9.17mg/L (target value above 3mg/L); the pH was 8.343-8.649 (target: not exceeding 6-9 and not vary by more than 1.5 units); the hardness was 150mg/L-165mg/L (as CaCO3), above 140mg/L (as CaCO3). For the medium at the beginning and during the limit test, the dissolved oxygen concentration was 7.96mg/L-8.25mg/L (target value above 3mg/L); the pH was 8.296-8.501 (target: not exceeding 6-9 and not vary by more than 1.5 units); the hardness was 145mg/L-155mg/L (as CaCO3), target value: above 140mg/L (as CaCO3).

10.2 Alga medium
The algae medium was used to culture the algae for feeding the test organisms in the test. Various analytical grade salts were dissolved in deionised water to obtain separate alga medium stock solutions. The alga medium were prepared by diluting the stock solutions in deionised water shortly before beginning of the test.

The Ingredients and their nominal concentrations of Elendt M4 Medium
The ingredients of Elendt M4 medium The nominal concentrations of the ingredients in the Elendt M4 medium
Stock solution I H3BO3 2859.5μg/L
MnCl2•4H2O 360.5μg/L
LiCl 306μg/L
RbCl 71μg/L
SrCl2•6H2O 152μg/L
NaBr 16μg/L
Na2MoO4 •2H2O 63μg/L
CuCl2•2H2O 16.75μg/L
ZnCl2 13μg/L
CoCl2•6 H2O 10μg/L
KI 3.25μg/L
Na2SeO3 2.19μg/L
NH4VO3 0.575μg/L
Na2EDTA•2H2O 250μg/L
FeSO4•7H2O 99.55μg/L
Stock solution II CaCl2•2H2O 293.8mg/L
MgSO4•7H2O 123.3mg/L
KCl 5.8mg/L
NaHCO3 64.8mg/L
Na2SiO3•9H2O 10mg/L
NaNO3 274μg/L
KH2PO4 143μg/L
K2HPO4 184μg/L
Vitamin stock solution Thiamine hydrochloride 75μg/L
Cyanocobalamine (B12) 1.0μg/L
Biotine 0.75μg/L




The Ingredients and their nominal concentrations of alga medium
The ingredients of alga medium The nominal concentrations of the ingredients in the stock solutions of the alga medium The nominal concentrations of the ingredients in the alga medium
Stock A NH4Cl 1.5g/L 15mg/L
MgCl2·6H2O 1.2g/L 12mg/L
CaCl2· 2H2O 1.8g/L 18mg/L
MgSO4·7 H2O 1.5g/L 15mg/L
KH2PO4 0.16g/L 1.6mg/L
Stock B FeCl3·6 H2O 64mg/L 64μg/L
Na2EDTA·2H2O 100mg/L 100μg/L
Stock C H3BO3 185mg/L 185μg/L
MnCl2·4 H2O 415mg/L 415μg/L
ZnCl2 3mg/L 3μg/L
CoCl2·6 H2O 1.5mg/L 1.5μg/L
CuCl2·2 H2O 0.01mg/L 0.01μg/L
Na2MoO4·2 H2O 7mg/L 7μg/L
Stock D NaHCO3 50g/L 50mg/L

Preparation of test solutions
Based on the information provided by the sponsor, the test substance is identified as an UVCB. Thus, the test solutions were prepared as water accommodated fraction (WAFs). WAFs were prepared by adding the required amount of the test substance into 2L Elendt M4. The mixtures were stirred for 1h in the dark. The vortex depth was 1/10 of the mixture column. After this time the test substance was visually completely dissolved. 1L mixture was siphoned from the middle portion of the solution in the vessel after being settled for one hour (the first 100mL were discarded). Before using in the test, the test solution was inspected by a microscope to ensure that the test substance was completely dissolved. The WAFs at each loading were prepared individually every three times a week.
Before using in the test, the pH of the highest treatment group measured was 8.418-8.665, not exceeding 6-9, no adjustment was needed.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnias (ID: DM-BSAL-F5-180502-M4) were originally supplied by MicroBioTests Inc. Young female Daphnia (the parent animals) aged less than 24 hours and not first brood progeny were used at the start of the test. Adult Daphnia magna were bred in the laboratory of APM in the Elendt M4 medium. The culture conditions were similar to test conditions. Cultures of adult Daphnia magna were fed concentrated freshwater suspensions of unicellular green algae Desmodesmus Subspicatus, Pseudokirchneriella subcapitata and Chlorella sp. at a rate equivalent to 0.1mg-0.2mg of organic carbon per adult per day.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

the hardness of the test solutions ranged from 145mg/L to 160mg/L (as CaCO3), and were thus above 140mg/L (as CaCO3)

pH:

During the test period, the pH values of the test solutions in the blank control group and the treatment group ranged from 8.183 to 8.501, and did thus not exceed the range 6-9 and did not vary by more than 1.5 units

Dissolved oxygen:

the dissolved oxygen concentrations of the test solutions ranged from 7.96mg/L to 8.79mg/L, and were thus above 3mg/L

Details on test conditions:

Range finding test:
Five treatment groups and one test-medium control were included in the range-finding test. The loading rates of the treatment group respectively were 0.256mg/L, 0.640mg/L, 1.60mg/L, 4.00mg/L and 10.0mg/L. Elendt M4 medium was used as a blank control group.
Ten animals individually held were tested for each test group. Each test loading rate group consisted of replicates filled with test solutions of the same loading rate. One animal was maintained individually in each replicate. The duration of the range-finding test was 21 days. The test vessels were not aerated during the test.
The range-finding test was performed in the illumination incubabor with airtight glass vessels and the ratio of light to dark was 16 hours to 8 hours. Parent animals were maintained individually, one per test vessel, with about 66mL of medium in each vessel.
A semi-static test was conducted. The frequency of medium renewal was three times a week in the range-finding test. The medium was renewed on Day 3, 5, 7,10, 12, 14, 17 and 19 during the test.
The measured concentrations of the test substance in each group were determined on Day 0, 1, 2 and 3 during the test. One test vessel was selected from each test group (i.e. replicate) randomly. Two 2mL test samples were taken respectively from each group and filtrated with 0.22μm cellulose nitrate membrane filters. One was used to analyze and the other was stored as a back up.

Procedure
Every adult was fed concentrated freshwater suspensions of unicellular green algae Desmodesmus Subspicatus, Pseudokirchneriella subcapitata and Chlorella sp. once a day at a rate equivalent to 0.15mg of organic carbon per adult per day.
The results of the observations made during the test were recorded once a day. The offspring produced by each parent animal were removed and counted from the appearance of the first brood. The numbers of live offspring, aborted egg and dead offspring were counted and recorded. Mortality among the parent animals was recorded at the same times as offsprings were counted.
During the test, when the medium was renewed in the semi-static test, a second series of test vessels had been prepared and the parent animals were transferred to them by a glass pipette of suitable diameter.

Results
Since the test substance is an UVCB, more than one peak was detected during the analysis. Six peaks were obtained by UPLC-MS analysis during the test, which may not necessarily represent the test substance completely. The peak area (%) of two main peaks (peak 1 and peak 2) were equal to or above 90% of the total peak area. Therefore and based on the study protocol, only peak 1 and peak 2 were quantified. The measured concentrations of peak 1 and peak 2 on Day 1, 2 and 3 in all treatment groups varied less than 20% compared with those on Day 0 (Section 19.5). Thus, the renewal frequency of the test solutions was three times a week during the test with renewal periods of up to 3 days.
The mortality of the parent animals in the blank control group and in the treatment groups were 0%, and did thus not exceed 20%.
According to the result of the Linear Interpolation (200 Resamples), the 50% reduction in reproductive output (EL50) was higher than 10mg/L. According to the result of the Dunnett's Test, the difference of 21 days reproduction between the treatment group of 10mg/L and the blank control was not statistically significant (p=0.34, p>0.05). The 21 days NOELR for reproduction of the test substance to Daphnia magna was higher than or equal to 10mg/L and the LOELR was higher than 10mg/L.
Based on the result of range-finding test, a limit test test was carried out.

Limit test
Preparation of test solutions
The test solutions were prepared as water accommodated fraction (WAFs). A loading rate of 10.0mg/L of the test substance was prepared by adding the required amount of the test substance into 2L Elendt M4. The mixtures were stirred for 1h in the dark. The vortex depth was 1/10 of the mixture column. After this time the test substance was visually completely dissolved. 1L mixture was siphoned from the middle portion of the solution in the vessel after being settled for one hour (the first 100mL were discarded). Before using in the test, the test solution was inspected by a microscope to ensure that the test substance was completely dissolved. The test solution was prepared three times a week.
Before using in the test, the pH of the treatment group measured was 8.183-8.425, not exceeding 6-9, no adjustment was needed.
Test methods
According to the results of range-finding test, one treatment group and one test-medium control were included in the limit test. The loading rate of the treatment group was 10.0mg/L.
For the limit test, ten animals individually held were tested for each test group. Each test loading rate group consisted of replicates filled with test solutions of the same loading rate. One animal was maintained individually in each replicate. The test duration was 21 days. The test vessels were not aerated during the test.
The limit test was performed in the illumination incubabor with airtight glass vessels and the ratio of light to dark was 16 hours to 8 hours. Parent animals were maintained individually, one per test vessel, with about 66mL of medium in each vessel.
A semi-static test was conducted. The frequency of medium renewal was three times a week in the limit test. The medium was renewed on Day 3, 5, 7, 10, 12, 14, 17, 19 of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The concentrations of the test substance in the test solutions were analyzed using UPLC-MS method. The measured concentrations of peak 1 and peak 2 in the treatment group varied less than 20% during each renewal period, even those of the freshly prepared solutions varied within ±20% of their geometric mean measured one, indicating that the concentration of the main components was kept stable .

The mortality of the parent animals in the blank control group and in the treatment group both were 0%, and did thus not exceed 20%.

The numbers of live offspring in each group during the test are given in section 19.7. The mean number of live offspring produced by each parent animal surviving at the end of the test in blank control group was 146, and exceeded thus the minimum required value of 60 and the coefficient of variation was 7.78%, and was thus not higher than 25%. According to the result of the Dunnett's Test, the difference of 21 days reproduction between the treatment group and the blank control was not statistically significant (p=0.95, p>0.05). The 21 days NOELR for reproduction of the test substance to Daphnia magna was higher than or equal to 10mg/L and the LOELR was higher than 10mg/L.
No aborted egg and dead offspring were observed in each group during the test.

The effect of Texapon SFA (BSCL20171115-2017) on Daphnia magna is as following:

Reproductive effect on Daphnia magna:
The 21 days No Observed Effect Loading Rate (NOELR) was higher than or equal to 10mg/L. The 21 days Lowest Observed Effect Loading Rate (LOELR) was higher than 10mg/L.

Length effect on Daphnia magna:
The 21 days No Observed Effect Loading Rate (NOELR) was higher than or equal to 10mg/L. The 21 days Lowest Observed Effect Loading Rate (LOELR) was higher than 10mg/L. The results above were expressed relative to the loading rate of the test substance, since it is not ensured that the effects were caused by the analyzed components.

Validity criteria fulfilled:

yes

Conclusions:

The 21 days No Observed Effect Loading Rate (NOELR) was higher than or equal to 10mg/L. The 21 days Lowest Observed Effect Loading Rate (LOELR) was higher than 10mg/L.

Executive summary:

After a range finding test, Daphnia magna were exposed in an OECD 211-test for 21 d towards a limit concentration of 10 mg/l test substance. The 21 days No Observed Effect Loading Rate (NOELR) was higher than or equal to 10mg/L. The 21 days Lowest Observed Effect Loading Rate (LOELR) was higher than 10mg/L.

Description of key information

After a range finding test, Daphnia magna were exposed in an OECD 211-test for 21 d towards a limit concentration of 10 mg/l test substance. The 21 days No Observed Effect Loading Rate (NOELR) was higher than or equal to 10mg/L. The 21 days Lowest Observed Effect Loading Rate (LOELR) was higher than 10mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

10 mg/L