Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-05 to 2015-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13th April, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The respective volumes of the aqueous samples (Level 1: 1.5 mL; level 2: 2 mL; level 3 0 4 mL; level 4: 0.2 mL; level 5 0.2 mL were diluted with copper free water ad 10 mL (level 1 3 and 4), 20 mL (level 5) or 25 mL (level 2), respectively.
- Sampling method: 0.5 mL of each dilution was mixed with 0.5 mL analytical grade methanol and measured by LC-MS/MS.
- Sample storage conditions before analysis: Measured directly.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest nominal concentration was prepared by adding the required nominal amount of the test item (200 mg/L test item) and stirring for at least 15 minutes at room temperature. To obtain the other required test concentration the highest assay was serially diluted with dilution water and thoroughly mixed, sampled for later chemical analysis and distributed to the test beakers. The test solutions were freshly prepared before test start.
- Controls: Promary controlwith K2Cr2O7 as reference and one further control consisted of dilution water only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: clone V
- Source: German Federal Environment Agency, lnstitut fi.'ir Wasser-, Boden und Lufthygiene. Specimens used in the test were bred in the laboratory of the Fraunhofer IME.
- Age of parental stock (mean and range, SD): 3 weeks old
- Feeding during test: no


ACCLIMATION
- Acclimation period: 1 week
- Acclimation conditions (same as test or not): no, room temperature in ca. 1.8 L dilution water
- Type and amount of food: algal suspension of Desmodesmus subspicatus
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

In spite of the passage through a lime-stone column, hardness was below the recommended (not required) range (140 - 250 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Historical data showed values of 70 — 120 mg/L as CaCO3. Total hardness: 1.1 mmol/L.

Test temperature:

18-22°C

pH:

7.6 - 8.3

Dissolved oxygen:

8.1 - 8.7 mg/L

Conductivity:

231 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 12.5, 25, 50, 100, 200 mg/L, recovery of the test substance was analytically determined and was out of the 80-120 % range, therefore the geometric means were calculated: 7.74, 19.6, 45.0, 94.6, and 196 mg/L a.s.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type: closed
- Material, size, headspace, fill volume: glass, 50 mL nominal volume, filled with about 50 mL test solution.
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates):4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a limestone column, and aeration.
- Total organic carbon: 0.41 mg/L
- Alkalinity: 1.6 mmol/L
- Ca/mg ratio: 1.0/0.1
- Conductivity: 231 µS/cm


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8
- Light intensity: 779-807 lx (corresponding to 10.3 — 10.8 µE/(m²s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The numbers of dead animals were visually determined daily and dead specimens (if occurred) were removed. Any abnormalities (if occurred) in appearance and behaviour were recorded. immobility was determined according to the OECD guideline 202. Specimens which were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilised (even if they were still moving their antennae).

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 196 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

94.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

- Mortality of control: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 24 h EC50 Value Immobilisation: 0.93 mg/L (95% CL: 0.83-1.04)

Reported statistics and error estimates:

For immobilisation, the NOEC and LOEC were determined. EC10, EC20, and EC50 values together with 95 % confidence intervals (where possible) were calculated by Probit-analysis assuming log-normal distribution of the values. All statistical calculations were performed using the computer programme ToxRat®.

NOEC and EC50 values for immobilisation at test end [mg a.s./L].

NOEC = No observed effect concentration; LOEC = Lowest obsen/ed effect concentration;

EC = Effect concentration; CL = Confidence level; n.d. = not determined due to mathematical

reasons; effect concentrations given as Qometric mean values.

	Mortality
NOEC	94.6
LOEC	196
EC10 (95% CL)	111 (n.d.)
EC20 (95% CL)	140 (n.d.)
EC50 (95% CL)	> 196

 

Validity criteria:

- With 0% the percent mortality observed in the controls over the test period is </= 10%.

- The dissolved oxygen concentration at the end of the aging period (48 h) was >/= 3 mg/L in control and test vessels.

Validity criteria fulfilled:

yes

Conclusions:

In the present study the NOEC and EC50 values in Daphnia magna for Rhamnolipids according to OECD Test giuideline 202 were determined. Since no mortality and no immobilisation occurred during the first 24 h of the test and immobilisation was only detected at the highest concentration applied after 48 h the substance was considered to be not acutely toxic to Daphnia magna until 94.6 mg/L (NOEC).

Executive summary:

The 48 hr-acute toxicity of Rhamnolipids to Daphnia magna was studied under static conditions according to PECD Guideline 202 (2004) and EU method C.2 (2008).  Daphnids were exposed to control, and test chemical at nominal concentrations of 12.5, 25, 50, 100, 200 mg/L (10.5, 21.0, 42.0, 84.0, and 168 mg a.i./L). Mortality/immobilization and sublethal effects were observed daily.

The mean measured test item concentrations of the freshly prepared test solution of the active substance (initial concentrations) of the test item were between 84.2 % and 116 % of nominal concentrations, and in the aged test solution between 64.6 % and 117 %. Due to recovery rates were not in a range of 80 - 120 % of nominal throughout the test, geometric means were applied for effect assessment. The geometric measured concentrations were 7.74, 19.6, 45.0, 94.6, and 196 mg a.s./.

No significantly increased mortality (NOEC) up to a concentration of 94.6 mg a.i./L was detected when compared to the control daphnids. The EC50 was estimated to be > 196 mg a.i./L.

 

Results Synopsis

Test Organism Age: Daphnia magna <24 h

Test Type: Static

EC50:  > 196mg a.i./L

Endpoint(s) Effected: mortality

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 196 mg/L

Additional information

The short-term screening-level data for aquatic toxicity determined EC50 (daphnids) > 196 mg/L.