O-6-deoxy-α-L-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl (2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate

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:

21/03/2018 - 11/04/2018

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

water

Details on test solutions:

Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared with 1 g of test item in 1 liter of medium. 6 à 8 bottles were prepared at the same time, every two days. After a period of stirring (about 24 hours) to obtain maximum solubility, the aqueous phase was collected using 0,45 μm filters. All bottles were filtered and mixed. Test solutions were prepared by dilution of this solution, every two days.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia, Daphnia magna Strauss clone 5, between 6 and 24 hours old and produced from parthenogenically reproducing brood female population were used. Daphnia are issued from the breeding lab. The juvenile stemming at least from the second litter were used to start the test.
Daily feeding: Pseudokirchneriella subcapitata and Chlorella vulgaris suspensions (65.106 cells/mL): about 10 mL/replicates/day

Test type:

flow-through

Water media type:

other: Elendt M4

Remarks:

The test medium used was Elendt-M4 medium. It was reconstituted with chemical reagents and deionised water. The test medium was performed several times a week.

Limit test:

no

Total exposure duration:

21 d

Hardness:

Hardness (CaCO3): Between 140 and 250 mg.L-1

Test temperature:

Temperature: 20 °C ± 2

pH:

pH: Between 6 and 9

Dissolved oxygen:

Between 8.2 and 9.3 mg/L

Details on test conditions:

Light intensity: between 1000 and 1500 lux with a light:dark cycle of 16 hours light and 8 hours dark.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

other: LC10survival

Effect conc.:

ca. 32.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 97.61 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

ca. 10.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 97.61 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 32.86 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 9.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

24-hour EC50 (mg.L-1) : 1.428
95% confidence limits (mg.L-1) : 1.348 – 1.509
Starting date 21 February 2018

Validity criteria fulfilled:

yes

Conclusions:

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).
Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.
Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared by agitation (about 24 hours). Test solutions were be prepared by dilution of this solution.
Test item quantification was performed according to a validated analytical method (final report RRCo-000146_01) using a LC-MS/MS from Thermo (LC Ultimate 3000 and MS/MS TSQ Quantiva) as mass spectrometry detection to measure O-6-deoxy-α-l-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl(2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate concentrations once a day in all concentrations. As some of the measured concentrations were not within  20% of nominal or initial concentrations, all results were based on mean measured concentrations.
The effects of the test item diluted in OECD test medium at the definitive measured concentrations of 0.43, 1.73, 9.13, 32.86 and 97.61 mg.L-1 to Daphnia magna were investigated under laboratory conditions.

Results are the following:
LC10 Survival (95% confidence limits) 32.9 mg.L-1 [3.76 mg.L-1 ; 93.9 mg.L-1]
LC50 Survival > 97.61 mg.L-1
EC10 Reproduction (95% confidence limits) 10.1 mg.L-1 [3.34 mg.L-1 ; 24.3 mg.L-1]
EC50 Reproduction > 97.61 mg.L-1
NOEC Survival 32.86 mg.L-1
NOEC Reproduction 9.13 mg.L-1

The study is valid since:
• the mortality of the parent animals (female Daphnia) was not exceed 20% at the end of the test.
• the mean number of living offspring produced per parent animal surviving at the end of the test was ≥ 60.
• EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L-1.
Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days exposure period were greater than 97.61 mg.L-1.

The lowest NOEC was estimated to be at 9.13 mg.L-1 corresponding to the NOEC Reproduction.
The flow-through rate was 1 L / day and the saturated solution was renewed every 2 days. The evacuation was by overflow. During the test, daphnia were fed ad libitum with alga generating a sedimentation and accumulation of algae in the test systems. As the test item could be adsorbed or metabolized by the algae (see final report OECD 201 RRCo-0000149_02), this could explain the decrease of measured concentration in the lower concentrations.
Consequently, exposure concentrations could be greater than the measured concentration (exposure by feeding and by test media) and so, NOEC values could be underestimated.

Executive summary:

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).

Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.

Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared by agitation (about 24 hours). Test solutions were be prepared by dilution of this solution.

Test item quantification was performed according to a validated analytical method (final report RRCo-000146_01) using a LC-MS/MS from Thermo (LC Ultimate 3000 and MS/MS TSQ Quantiva) as mass spectrometry detection to measure O-6-deoxy-α-l-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl(2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate concentrations once a day in all concentrations. As some of the measured concentrations were not within  20% of nominal or initial concentrations, all results were based on mean measured concentrations.

The effects of the test item diluted in OECD test medium at the definitive measured concentrations of 0.43, 1.73, 9.13, 32.86 and 97.61 mg.L-1 to Daphnia magna were investigated under laboratory conditions.

 

Results are the following:

LC10 Survival (95% confidence limits) 32.9 mg.L-1 [3.76 mg.L-1 ; 93.9 mg.L-1]

LC50 Survival > 97.61 mg.L-1

EC10 Reproduction (95% confidence limits) 10.1 mg.L-1 [3.34 mg.L-1 ; 24.3 mg.L-1]

EC50 Reproduction > 97.61 mg.L-1

NOEC Survival 32.86 mg.L-1

NOEC Reproduction 9.13 mg.L-1

 

The study is valid since:

• the mortality of the parent animals (female Daphnia) was not exceed 20% at the end of the test.

• the mean number of living offspring produced per parent animal surviving at the end of the test was ≥ 60.

• EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L-1.

Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days exposure period were greater than 97.61 mg.L-1.

 

The lowest NOEC was estimated to be at 9.13 mg.L-1 corresponding to the NOEC Reproduction.

The flow-through rate was 1 L / day and the saturated solution was renewed every 2 days. The evacuation was by overflow. During the test, daphnia were fed ad libitum with alga generating a sedimentation and accumulation of algae in the test systems. As the test item could be adsorbed or metabolized by the algae (see final report OECD 201 RRCo-0000149_02), this could explain the decrease of measured concentration in the lower concentrations.

Consequently, exposure concentrations could be greater than the measured concentration (exposure by feeding and by test media) and so, NOEC values could be underestimated.

Description of key information

The objective of this study was to assess the effects of the test item on the reproductive output of Daphnia magna over a period of 21 days according to OECD 211 guideline (2 october 2012).
Daphnia magna is a freshwater crustacean that is recommended by the OECD Guideline 211.
Since the test item is poorly soluble under the test conditions, a saturated solution of test item (limit of solubility LS) was prepared by agitation (about 24 hours). Test solutions were be prepared by dilution of this solution.
Test item quantification was performed according to a validated analytical method (final report RRCo-000146_01) using a LC-MS/MS from Thermo (LC Ultimate 3000 and MS/MS TSQ Quantiva) as mass spectrometry detection to measure O-6-deoxy-α-l-mannopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl(2α,3β,4α)-2,3,23-trihydroxyurs-12-en-28-oate concentrations once a day in all concentrations. As some of the measured concentrations were not within  20% of nominal or initial concentrations, all results were based on mean measured concentrations.
The effects of the test item diluted in OECD test medium at the definitive measured concentrations of 0.43, 1.73, 9.13, 32.86 and 97.61 mg.L-1 to Daphnia magna were investigated under laboratory conditions.

Results are the following:
LC10 Survival (95% confidence limits) 32.9 mg.L-1 [3.76 mg.L-1 ; 93.9 mg.L-1]
LC50 Survival > 97.61 mg.L-1
EC10 Reproduction (95% confidence limits) 10.1 mg.L-1 [3.34 mg.L-1 ; 24.3 mg.L-1]
EC50 Reproduction > 97.61 mg.L-1
NOEC Survival 32.86 mg.L-1
NOEC Reproduction 9.13 mg.L-1

The study is valid since:
• the mortality of the parent animals (female Daphnia) was not exceed 20% at the end of the test.
• the mean number of living offspring produced per parent animal surviving at the end of the test was ≥ 60.
• EC50 with reference item over a period of 24 hours must be between 0.6 and 2.1 mg.L-1.
Based on the reproduction rate and the survival rate, the EC50 and LC50 after a 21 days exposure period were greater than 97.61 mg.L-1.

The lowest NOEC was estimated to be at 9.13 mg.L-1 corresponding to the NOEC Reproduction.
The flow-through rate was 1 L / day and the saturated solution was renewed every 2 days. The evacuation was by overflow. During the test, daphnia were fed ad libitum with alga generating a sedimentation and accumulation of algae in the test systems. As the test item could be adsorbed or metabolized by the algae (see final report OECD 201 RRCo-0000149_02), this could explain the decrease of measured concentration in the lower concentrations.
Consequently, exposure concentrations could be greater than the measured concentration (exposure by feeding and by test media) and so, NOEC values could be underestimated.