N-(2-hydroxyethyl)stearamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 07, 2014 to March 06, 2015

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:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10706

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, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the first test samples for analysis were taken from the test concentration and the control. In addition, the filter containing undissolved residue was kept for possible analysis.
- Frequency: At the beginning and at the end of three intervals of 48 h (nominal Days 0 and 2, 6 and 8, 14 and 16).
- Volume: 2.0 mL
- Storage: Not applicable, samples were analysed on the day of sampling.
At the end of the refreshment period, the replicates were pooled at each concentration before sampling.
No samples for analytical confirmation of actual exposure concentrations were taken during the second test.

Vehicle:

yes

Details on test solutions:

PREPARATION OF SOLUTIONS:
Stock and spiking solutions
Stock solutions of the test substance were prepared in methanol at concentrations of 2000–3000 mg/L. In order to dissolve the test substance the solutions were ultrasonicated for 15 minutes.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was methanol.

PREPARATION OF TEST SOLUTIONS:
All loading rates ≥1.0 mg/L were prepared separately applying a 10 minute treatment period with ultrasonic waves followed by 1 d of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were filtered through a 0.45 μm membrane filter (Whatman) where after the clear and colourless Water Accommodated Fractions (WAFs) were used for testing. For the range-finding test one lower concentration was prepared by a ten-fold dilution of the 1.0 mg/L WAF.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Age of parental stock: Two weeks old.
- Feeding during test: Twice daily an amount of 0.25 mL of Chlorella pyrenoidosa 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, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211. From Day 15 onwards, the total daily amount was increased to 0.75 mL, based on expertise. This daily ration corresponded to 0.3 mg C/Daphnia/day.

ACCLIMATION
- Acclimation conditions: Same
- Type and amount of food: Daily, a suspension of fresh water algae

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

- First reproduction test: 179 to 232 mg CaCO3 per liter
- Second reproduction test: 196 to 214 mg CaCO3 per liter

Test temperature:

- First reproduction test: between 18.1 and 20.7°C
- Second reproduction test: between 19.2 and 20.4°C

pH:

- First reproduction test: 7.9 to 8.5
- Second reproduction test: 7.8 to 8.8

Dissolved oxygen:

- First reproduction test: 8.5 to 10.1 mg/L
- Second reproduction test: 8.7 to 10.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Volume first test: 80 mL, all-glass covered with a Perspex plate, Volume second test: 60 mL, all-glass covered with a Perspex plate
- Renewal duration of test solution: Every 48 h
- No. of organisms per vessel:First test: At the start of the experiment (nominal Day 0) 20 neonate daphnids, less than 1 d old, per group were divided over twenty vessels each containing a minimum of 50 mL test medium.
Second test: At the start of the experiment (nominal Day 0) 10 neonate daphnids, less than 1 d old, per group were divided over ten vessels each containing a minimum of 50 mL test medium. The control group consisted of 20 daphnids.
- Loading rate: First reproduction test: Control (0) and 100 mg/L, Second reproduction test: Control (0), 1, 10 and 100 mg/L

TEST MEDIUM / WATER PARAMETERS
- Total organic carbon: - First reproduction test: between 18.1 and 20.7°C
- Second reproduction test: between 19.2 and 20.4°C
- Medium: M7, as prescribed by Dr. Elendt-Schneider

OTHER TEST CONDITIONS
- Photoperiod: First test: 16 h photoperiod daily;
Second test: 16 h photoperiod daily;

- Light intensity: First test: intensity at the start: 524-598 lux, intensity at the end: 530-552 lux
Second test: intensity at the start: 675-747 lux, intensity at the end: 689-763 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parental daphnids
- Condition: Every workday and upon renewal on non-workdays, 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 and upon renewal on non-workdays.
- Body length: At the end of the test.

Offspring
- Appearance first brood: When observed.
- Newborn daphnids: Every workday and upon renewal on non-workdays, 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 medium
Temperature, oxygen and pH At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids.

RANGE-FINDING STUDY
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No mortality was observed at any of the WAFs and the control. Most surviving daphnids in the control and the respective WAFs carried eggs in their brood pouch on Day 7. Therefore, no effects on survival or time of egg development were observed at any of the WAFs when compared to the control.
The analytical results show that the actual test concentrations are quite low when detected (~0.1-1.0 μg/L) and approximately the same for all WAFs. Concentrations could not be detected in the old solutions. However, this seems to be partly related to the presence of algae.
Test conditions during the range-finding test were maintained within the limits prescribed by the protocol.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: The effect concentration is expressed in terms of loading rate.

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: The effect concentration is expressed in terms of loading rate.

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: The effect concentration is expressed in terms of loading rate.

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: The effect concentration is expressed in terms of loading rate.

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: The effect concentration is expressed in terms of loading rate.

FIRST REPRODUCTION TEST

Measured concentrations

The analytical results showed that the actual test concentration in the WAF was quite low and ranged between 0.48 and 4.4 μg/L. The analytical method did not allow the determination of what constituent(s) of the UVCB were in solution. Furthermore, the concentrations measured were below the calibration curve and therefore had to be estimated. This explains the variation in concentration at the given time points. Unfortunately the method cannot be further adjusted for the measurement of more accurate concentrations. Therefore, the mean exposure concentration was calculated from all concentrations measured. This resulted in a mean concentration of 1.2 μg/L.

The analytical results were not in agreement with the given water solubility of 75 mg/L. In a GLP test performed at WIL Research the water solubility of the test substance was determined to be <1.0 mg/L (Project 499359), which was more in agreement with the current results.

Condition of parental daphnids

Two out of the twenty parental daphnids died during the test period in the control. Hence, parental mortality did not exceed 20% in the controls. Parental mortality in the test concentration was identical (two out of twenty). Hence, parental mortality in the test concentration was statistically not significantly different when compared to the control.

In the control, the presence of eggs in the brood pouch was recorded for the first time on Day 7 and the first brood appeared on Day 10. The first recording of the presence of eggs in the brood pouch was approximately 1 d later in the test concentration than in the control. Hence, a slight delay in appearance of the first brood was observed in the test concentration when compared to the control.

Reproduction

On average, 112 juveniles were produced per surviving daphnid in the control. Statistically significant reduction of reproduction was found in the test concentration. A large variation in reproduction between replicates was observed for the test concentration.

Based on the above results, it is implied that the increasing number of immobile offspring in the test concentration was related to the test substance.

Body length

Statistically significant reduction of growth was found in the test concentration compared to the control.

SECOND REPRODUCTION TEST

Condition of parental daphnids

None out of the twenty parental daphnids died during the test period in the control. Hence, parental mortality did not exceed 20% in the controls. Parental mortality in the test concentrations was 0-10%. Hence, parental mortality in the test concentrations was statistically not significantly different when compared to the control.

In the control, the presence of eggs in the brood pouch was recorded for the first time on Day 5/6 and the first brood appeared on Day 8/9. The first recording of the presence of eggs in the brood pouch was approximately 1 d later in the test concentration than in the control. Hence, a slight delay in appearance of the first brood was observed in the test concentration when compared to the control.

Reproduction

On average, 92 juveniles were produced per surviving daphnid in the control. Statistically significant reduction of reproduction was found in all three test concentrations. A large variation in reproduction between replicates was observed for the test concentrations.

Based on the above results, it is implied that the increasing number of immobile offspring in the test concentration was related to the test substance.

Body length

Statistically significant reduction of growth was found in all three test concentrations.

Determination of effect concentrations

It was not possible to accurately prepare loading rates below 0.2 -1.0 mg/L. Unfortunately, the NOEC or in this case the NOELR was below the lowest loading rate of 1.0 mg/L and therefore no exact value for the NOEC/NOELR could be given. Since the WAF procedure proved also to be inadequate for generating test solutions there was no option left for the preparation of appropriate test concentrations.

Further investigation

The turbidity values of the freshly prepared 0.2 mg/L WAF in ISO and in M7 medium were 0.140 and 0.209 NTU (Nephelometric Turbidity Units), respectively. The turbidity values of the freshly prepared 1.0 mg/L WAF in ISO and in M7 medium were 0.148 and 0.314 NTU, respectively. The turbidity values of the freshly prepared 10 mg/L WAF in ISO and in M7 medium were 3.02 and 3.20 NTU, respectively. <0.1 NTU is considered as clear. The turbidity of the analytical blanks (ISO and M7-medium with no test substance) ranged between 0.207 and 0.263 NTU.

Based on the turbidity measurements only the WAF prepared at a loading rate of 0.2 mg/L did not contain significant amounts of undissolved test substance, when using M7 medium.

Validity criteria fulfilled:

yes

Conclusions:

The test substance significantly reduced the reproduction of Daphnia magna in a WAF prepared at a loading rate of 1.0 mg/L after 21 d of exposure. Hence, the NOELR was <1.0 mg/L.

Executive summary:

A study was conducted to assess the long-term toxicity of the test substance, C16-18 MEA, to Daphnia magna according to OECD Guideline 211, ISO International Standard 10706 and EU Method C.20, in compliance with GLP. The substance was not completely soluble in test medium at the loading rates initially prepared. Hence, all test solutions were prepared separately, the use of solvents was omitted as the test substance was an UVCB and a Water Accommodated Fraction (WAF) procedure was followed. Two reproduction tests were performed under semi-static conditions, based on the results of a 7 d range-finding study. The first test, run as a limit test, included 20 vessels for the test concentration (WAF prepared at a loading rate of 100 mg/L) and 20 vessels for a control group. As effects were noted in the first study, a second reproduction test was run, including 10 vessels for the test concentrations (WAFs prepared at loading rates of 1.0, 10 and 100 mg/L) and 20 vessels for a control group. Each of the vessels contained one neonate (<24 h old) Daphnia magna and 50 mL of test medium. The test duration was 21 d and the test solutions were renewed every 48 h. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every workday and upon renewal on non-workdays, the condition of the parental daphnids was recorded. During the reproduction phase, the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test, the length of the surviving parental daphnids was measured. During the first reproduction test, samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of three intervals of 48 h. The analytical results showed that the actual test concentration in the WAF at a loading rate of 100 mg/L ranged between 0.48 and 4.4 μg/L. The analytical method did not allow the determination of what constituent(s) of the UVCB were in solution. Furthermore, the concentrations measured were below the calibration curve and therefore had to be estimated, causing a variation in measured concentrations at given time points. The method could not be further adjusted for the measurement of more accurate concentrations. Therefore, the mean exposure concentration was calculated from all concentrations measured. This resulted in a mean concentration of 1.2 μg/L. Given the above limitations, the analytical method was considered unreliable. Hence the results were expressed based on nominal loading rates rather than mean measured concentrations and no samples for analytical confirmation of actual exposure concentrations were taken during the second reproduction test. In the first reproduction test, parental mortality did not exceed 20% in the controls (two out of twenty). Parental mortality in the test concentration was identical (two out of twenty). Hence, parental mortality in the test concentration was statistically not significantly different when compared to the control. However, the exposure to the test substance was characterised by a slight delay in appearance of the first brood, statistically significant reduction of reproduction and growth was found in the test concentration compared to the control. In the second reproduction test, no parental mortality was recorded in the controls. Parental mortality in the test concentrations was 0-10%. Hence, parental mortality in the test concentrations was not significantly different when compared to the control. Similar to the limit test, a slight delay in appearance of the first brood was observed in the test concentration when compared to the control. Statistically significant reduction of reproduction was found at all three test concentrations. A large variation in reproduction between replicates was observed for the test concentrations. Statistically significant reduction of growth was also recorded in all three test concentrations. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test substance significantly reduced the reproduction of Daphnia magna in a WAF prepared at a loading rate of 1.0 mg/L after 21 d of exposure. Hence, the NOELR was <1.0 mg/L (Bouwman, 2015).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-2021 (testing planned)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Test organisms (species):

Daphnia magna

Water media type:

freshwater

Total exposure duration:

21 d

Reference substance (positive control):

no

Remarks on result:

other: Testing planned

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L

Additional information

A study was conducted to assess the long-term toxicity of the test substance, C16-18 MEA, to Daphnia magna according to OECD Guideline 211, ISO International Standard 10706 and EU Method C.20, in compliance with GLP. The substance was not completely soluble in test medium at the loading rates initially prepared. Hence, all test solutions were prepared separately, the use of solvents was omitted as the test substance was an UVCB and a Water Accommodated Fraction (WAF) procedure was followed. Two reproduction tests were performed under semi-static conditions, based on the results of a 7 d range-finding study. The first test, run as a limit test, included 20 vessels for the test concentration (WAF prepared at a loading rate of 100 mg/L) and 20 vessels for a control group. As effects were noted in the first study, a second reproduction test was run, including 10 vessels for the test concentrations (WAFs prepared at loading rates of 1.0, 10 and 100 mg/L) and 20 vessels for a control group. Each of the vessels contained one neonate (<24 h old) Daphnia magna and 50 mL of test medium. The test duration was 21 d and the test solutions were renewed every 48 h. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every workday and upon renewal on non-workdays, the condition of the parental daphnids was recorded. During the reproduction phase, the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test, the length of the surviving parental daphnids was measured. During the first reproduction test, samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of three intervals of 48 h. The analytical results showed that the actual test concentration in the WAF at a loading rate of 100 mg/L ranged between 0.48 and 4.4 μg/L. The analytical method did not allow the determination of what constituent(s) of the UVCB were in solution. Furthermore, the concentrations measured were below the calibration curve and therefore had to be estimated, causing a variation in measured concentrations at given time points. The method could not be further adjusted for the measurement of more accurate concentrations. Therefore, the mean exposure concentration was calculated from all concentrations measured. This resulted in a mean concentration of 1.2 μg/L. Given the above limitations, the analytical method was considered unreliable. Hence the results were expressed based on nominal loading rates rather than mean measured concentrations and no samples for analytical confirmation of actual exposure concentrations were taken during the second reproduction test. In the first reproduction test, parental mortality did not exceed 20% in the controls (two out of twenty). Parental mortality in the test concentration was identical (two out of twenty). Hence, parental mortality in the test concentration was statistically not significantly different when compared to the control. However, the exposure to the test substance was characterised by a slight delay in appearance of the first brood, statistically significant reduction of reproduction and growth was found in the test concentration compared to the control. In the second reproduction test, no parental mortality was recorded in the controls. Parental mortality in the test concentrations was 0-10%. Hence, parental mortality in the test concentrations was not significantly different when compared to the control. Similar to the limit test, a slight delay in appearance of the first brood was observed in the test concentration when compared to the control. Statistically significant reduction of reproduction was found at all three test concentrations. A large variation in reproduction between replicates was observed for the test concentrations. Statistically significant reduction of growth was also recorded in all three test concentrations. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test substance significantly reduced the reproduction of Daphnia magna in a WAF prepared at a loading rate of 1.0 mg/L after 21 d of exposure. Hence, the NOELR was <1.0 mg/L (Bouwman, 2015).

Also,after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a long-term toxicity to aquatic invertebrates study according to OECD Guideline 211 is planned with C16-18MEA in order to obtain information on the substance itself and further support the read across approach proposed for the FAA category members.