Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

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:

March 01 - June 07, 2017

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 (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch Number: E6.2-F04
- Purity, including information on contaminants, isomers, etc.: Purity: 90.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature, in a cool and well ventilated place. Keep container dry and closed.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Compatible material fosr storage containers: Glass; Metal or Polyethylene.

Analytical monitoring:

yes

Remarks:

The concentrations of the test item in the test media were determined by Ultra Performance Liquid Chromatography coupled with tandem Mass Spectrometer (UPLC-MS/MS).

Details on sampling:

Samples of fresh test solutions were taken from the test solution preparation just before distributing it to the ten replicates. For aged test solutions samples were taken from a representative test vessel per concentration. If analysis could not be applied immediately after sampling, samples were stored under circumstances to maintain the test item stable to the best of knowledge until analysis

Vehicle:

yes

Remarks:

water

Details on test solutions:

The test item solutions were prepared in dilution water. Initially, a stock solution was prepared by weighing in 220.26 mg test item into 2-liter Cu-free dilution water (test start - day 7) or 110.13 mg test item into 1 liter Cu-free dilution water (day 7 - test end) and stirring it for at least 30 minutes at room temperature. The stock solution served as the highest test concentration. The other required test solutions were prepared by adding 9.5, 30.5, 97.7, and 312.5 mL of the stock solution into Cu-free dilution water, filling it up to one liter. Afterwards the test solutions were mixed thoroughly again and were stirred for 5 -10 minutes. After preparation of test solutions samples were taken for chemical analysis and then the test solutions were filled into the test vessels

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Amgna
- Strain/clone: Daphnia magna
- Age at study initiation: 4 - 24 hours
- Valve height at study initiation, for shell deposition study (mean and range, SD):
- Peripheral shell growth removed prior to test initiation:
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene.
- Age of parental stock (mean and range, SD):
- Feeding during test: The daphnids were fed during the test with suspensions of unicellular alga Desmodesmus subspicatus
- Food type: a mixture of suspensions of unicellular alga Desmodesmus subspicatus. The content of food in the test suspensions, measured at 585 nm, was kept constant at 0.2 mg C/(Daphnia*day).



ACCLIMATION
- Test course: The beakers were covered with glass panes and subjected to a light/dark cycle of 16/8 hours. The test temperature during the test was 18 - 22°C. Temperature did not vary by more than 2°C within these limits. The light intensity did not exceed 15 -20 μE/(m2*s) or 1125 - 1500 lux.
- Type and amount of food: The daphnids were fed during the test with a mixture of suspensions of unicellular alga Desmodesmus subspicatus. The content of food in the test suspensions, measured at 585 nm, was kept constant at 0.2 mg C/(Daphnia*day).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Hardness was checked once a week at water renewal in new and aged control media and test solutions of the highest treatment.

Test temperature:

18 - 22°C. Was checked directly before adding the animals and at each water renewal in new and aged test solutions

pH:

7.13 – 8.93. Was checked directly before adding the animals and at each water renewal in new and aged test solutions

Dissolved oxygen:

Was checked directly before adding the animals and at each water renewal in new and aged test solutions

Nominal and measured concentrations:

Nominal concentrations: 1.05, 3.36, 10.76, 34.42, and 110.13 mg test item (TI)/L, corresponding to 0.95, 3.05, 9.77, 31.25 and 100.0 mg active ingredient (a.i)./L (measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: Numbered glass beakers (50 mL). The vessels were filled up with
about 50 mL test solution.
- Type: The beakers were covered with glass panes to prevent from evaporation as much as possible, but also permit gaseous exchange between the mediumand the atmosphere and access of light.
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
Cu-Free

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 16/8 hours.
- Light intensity: 15 -20 μE/(m2*s) or 1125 - 1500 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility, Reproduction, Lenght, Intrinsic rate r

Reference substance (positive control):

yes

Remarks:

The sensitivity of the test clone was checked twice a year in non-GLP tests by using K2Cr2O7 as reference substance. K2Cr2O7 is a recommended reference substance for acute effects on daphnids according to the OECD guideline 202

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

42.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

34.27 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: A significant difference in the length of individuals in the control and at least one treatment was also shown. Since there were no living individuals at
test end in the highest concentration, the length could just be determined for the control and the four lower test concentrations.
- Other biological observations: No other clinical signs were observed in any replicate at any concentration tested. Neither any physical nor pathological symptoms were obtained. Adult body length showed significant differences between control and the two highest treatments
- Mortality of control: There was a significant difference in survival of individuals between control and the highest treatment with test item. Thus, a concentration relating mortality of the adults was observed in the treatment with the test item.
- Reproduction and population growth: Age at the first brood was between 8.9 and 10.2 days for control and the treatments. The difference was not statistically significant. There was no reproduction in the highest treatment at all.
The cumulative number of offspring per introduced adult Daphnia ranged from 67.3 to 83.9 in
the control and the four lower treatments with test item but there was no offspring in the highest treatment. The cumulative number of offspring per survived adult Daphnia ranged from 67.3 to 86.6 in the control and the four lower treatments with test item. The difference of the number of offspring between the control and the highest treatment was statistically significant.
The intrinsic rate of increase ranged from 0.320 in the control to 0.289 in the second highest
treatment. The difference of the intrinsic rate between control and the second highest
treatment was statistically significant. Thus, they suggest a test item depending effect. The
highest concentration was excluded of this evaluation since there was no reproduction.
Since there was a significant trend in immobility of individuals in the control and the highest
treatment it was required by the statistic program to evaluate the results based on the cumulative offspring per introduced adult since the observed parental mortality was a dose dependent effect of the test substance and these values are lower than in the cumulative offspring per survivor.
There were significant, concentration-dependent effects on the reproduction and immobility of Daphnia magna over the range of the tested concentrations as well as on the length of the daphnids and the intrinsic rate . Effects on the age of the first reproduction were not significantly different from the control.

Effective concentrations: Based on the time weighted mean concentrations the EC10 for reproduction per introduced Daphnia magna was calculated to be 34.27 mg a.i./L and the respective EC50 was calculated to be 38.61 mg a.i./L. For immobility a LC10 of 11.26 mg a.i./L and an LC50 of 30.48 mg a.i./L was calculated.
Due to a lacking concentration response for the length no EC could be calculated.
For the intrinsic rate r an EC10 of 37.91 mg a.i./L was determined and the EC50 was above the
highest test concentration and therefore extrapolated to be 1112.77 mg a.i./L.
The NOEC for reproduction and immobility was calculated to be at 18.97 mg a.i./L based on
time weighted mean concentrations of test item. The NOEC for the intrinsic rate r was determined to be at 5.16 mg a.i./L. The NOEC for age of first reproduction and development rate seems to be ≥ 18.7 mg a.i./L. A NOEC for length could not be determined.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Relevant effect levels: 24 h EC50 value: Immobilization: 1.088 mg/L (95% CL: 0.632 – 1.841 mg/L)

Reported statistics and error estimates:

The following endpoints observed in the reproduction test are evaluated quantitatively:
 Mortality (Immobility) of parental generation daphnids
 Time to the first brood
 Cumulative number of live offspring at day 21 (study end)
 Individual length of adults
The evaluation is based on the time weighted mean measured test item concentrations

Evaluation and statistics: 

• At the end of the test, the total number living offspring produced per parent daphnia were counted excluding the number of offspring in the analysis from parent daphnids which died accidentally and/or inadvertently during the test.


• For offspring number, the total number of living offspring produced at the end of the test per parent Daphnia at the start of the test were compared.


• The parental mortality, time to first brood and offspring number were used to calculate the intrinsic rate of population increase r as integrative parameter relevant for population effects.


• The deviation from the mean measured test item concentrations was greater than ± 20 % of nominal at test end and therefore the analysis of the results were based on the time weighted mean measured active test substance (a.i.) concentrations.


• For each endpoint, the NOEC and LOEC, and, if possible, the EC50 and EC10 was
  determined. A LOEC was calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test. no effect was observed up to the highest tested concentration the NOEC values were determined using appropriate statistical methods.


• Since the test results of the reproduction test show a concentration-response relationship they were statistically analyzed to determine an EC50, EC20 and EC10 value together with 95 % confidence intervals using Probit-analysis assuming log-normal distribution of the values.


• Statistical calculations were done with the computer program ToxRat® Pro 3.2.1, a
  statistical software package for ecotoxicity response analysis by ToxRat® Solutions.

RESULTS

RESULTS

Test concentrations: The concentrations of the active test substance (a.i.) in the test media were determined by chemical analysis in the aqueous phase of all treatment levels. Active test substance (a.i.) concentrations were measured by Ultra-Perfomance Liquid Chromatography (UPLC) coupled to tandem Mass Spectrometry (MS/MS) at test start, after each media renewal and at the test termination of the daphnia test (LOQ = 0.2 mg/L).

 

Details of the analytical method and results are presented in Attachement to overall remarks. The test item solutions were prepared in dilution water. The stock solution served as the highest test concentration. The other required test solutions were prepared by adding 9.5, 30.5, 97.7, and 312.5 mL of the stock solution into Cu-free dilution water and filling it up to one liter, resulting in five test concentrations with a arithmetic mean measured test item concentration of freshly prepared test solutions of 54.6 – 93.8 % of nominal concentrations.

During the time interval until renewal of the test solution, test item concentrations decreased to 21.8 – 69.9 % of nominal.

Since the deviation of the mean measured test concentration was greater than ±20% of nominal concentrations, the evaluation of the effects was performed on basis of the time weighted mean test concentrations which were calculated for the evaluation of the biological parameters and the endpoints LOEC/NOEC. For concentrations below the LOQ, a value half of the LOQ was set.

The time weighted mean is the total area divided by the total days.

The time weighted mean of mean measured initial and mean measured aged concentration at test solution renewal were 0.32, 1.38, 5.16, 18.97 and 77.74 mg a.i./L, corresponding with 33.6, 45.2, 52.8, 60.7 and 77.7 % of the nominal concentrations. The deviation from the nominal test concentration was greater than 20 % and therefore the evaluation of the results was based on the time weighted mean concentrations.

Validity criteria fulfilled:

yes

Remarks:

Mortality in controls did not exceed 20%; the mean number of offspring in the control within the 21 days (79.7) was above the criterion of 60 / female; the DOC was above 3 mg/l at the beginning and during the test; The pH was 6 - 9;

Conclusions:

Reproduction as well as survival of the introducted partens was affected at the highest test item concentration of 77.74 mg a.i / L (LOEC), corresponding to 85.62 mg test item/L. the EC10 and EC50 for reproduction was calculated to be 34.27 ng a.i./L and 38.33 mg a.i./L, corresponding to 37.74 mg TI/L and 42.21 mg TI/L. For Immobility the
calculated LC10 was 11.64 mg a.i./L and the LC50 was 30.48 mg a.i./L, corresponding to 12.82 mg TI/L and 33.57 mg TI/L. A significant effect could also be observed for the length and the intrinsic rate r. Thus, the test item has a chronic adverse effect on reproduction of adult Daphnia magna under the chosen test conditions.

Description of key information

A semi-static reproduction test with Daphnia magna was conducted according to OECD 211 in order to investigate the chronic effects of the test item on daphnids. In the present study, five nominal concentrations (1.05, 3.36, 10.76, 34.42, and 110.13 mg test item (TI)/L, corresponding to 0.95, 3.05, 9.77, 31.25 and 100.0 mg active ingredient (a.i)./L (measured concentrations). The EC10 was determined to be 34.27 mg/L, based on the reproduction.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

ca. 34.7 mg/L