Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-27 to 2015-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Concentrations of the test item within the test medium were: 7.89, 17.4, 38.2, 84.0 and 185 mg a.s./L.
- Sampling method: A respective volume of the aqueous samples (Level 1: 2 mL; level 2: 1 mL; level 3: 0.4 mL; level 4: 0.2 mL; level 5: 0.2 mL) was diluted with copper free water ad 10 mL (level 1 through 4) or 25 mL (level 5), respectively.
- Sample storage conditions before analysis: Samples were measured directly.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All stock solutions and the medium were prepared with purified water processed using an ELGA "PURELAB Ultra“. The pH of the medium was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.
- Controls: growth medium control

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Gottingen, Albrecht von Haller Institut, Untere Klarspiile 2, D-37073 Gottingen, Catalog No 61.81 SAG
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same as in test
- Any deformed or abnormal cells observed: Not observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2°C

pH:

at the beginning 8.06 - 8.4 and at termination 8.00 - 8.75

Dissolved oxygen:

no specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

Nminal Control, 9.39, 20.7, 45.5, 100 and 220 equivalent to Control, 7.89, 17.4, 38.2, 84.0 and 185 mg a.s./L (based on 84 % active substance content)

Details on test conditions:

TEST SYSTEM
- Test vessel: The test vessels used were 250 mL conical glass flasks covered with air-permeable silicone- sponge caps.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 mL fill volume
- Initial cells density: 1.139E6 cells/mL
- Control end cells density: 1.139E6 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: permanent light; (day light: OSRAM “day light”) adjusted to 60 - 120 pmol/(m²s) (4440-8880 Iux)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations: 0.1, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: Compared to control there was no inhibition of yield and growth rate up to the highest test concentration of 10 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 220 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 220 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 220 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 2.58 mg/L (95% confidence limits: 2.48 - 2.68 mg/L), validity range 3.38 ± 1.30 mg/L

Reported statistics and error estimates:

Since the test results of the growth inhibition test (exposure period) showed no inhibition around 50 % they were not statistically analysed to determine an EC50, EC20 and EC10 value together with 95 % confidence intervals. NOEC values were determined using appropriate statistical methods (e.g. ANOVA followedby Williams‘ Multiple Sequential t-Test .

Table 1: Growth rate (G) and its inhibition relative to control (% I) as computed from the raw data for growth test intervals selected

Treatment, test item nominal [mg/L]	0-24		0-48		0-72
	G	% I	G	% I	G	% I
control	2.063	-	1.875	-	1.850	-
9.39	2.138	-3.61	1.941	-3.49	1.824	1.39
20.7	2.186	-5.97	1.954	-4.18	1.849	0.02
45.5	2.172	-5.28	2.094	-11.38	1.906	-2.10
100	2.226	-7.90	2.089	-11.38	1.906	-3.03
220	2.463	-19.4	2.201	-17.40	1.937	-4.73

Validity criteria:

- The cell number in the control cultures increased by a factor of 257 within the 72 hour test period (validity criterion: > 16).

- Evaluation of the sectional growth rates of the controls: The mean of the replicate coefficients of variations (CV %) in the section-by-section growth rate of controls was 12 % during the test period (validity criterion</= 35 %).

- The coefficient of variation of average specific growth rate at test end in replicate control cultures was 1.1% (validity criterion </=7%).

Validity criteria fulfilled:

yes

Conclusions:

The present study was conducted to determine the acute toxicity (within 72 h) of Rhamnolipids to freshwater algae. Acute toxicity is detected by inhibition of growth and yield of Pseudokirchneriella subcapitata. Since there was no sign of growth or yield inhibition, the test item is considered to be not actue toxic to Pseudokirchneriella subcapitata. The calculated NOEC is therefore > 220 mg/L.

Executive summary:

In a 72 hour acute toxicity study according to OECD Guideline 201 (2011), the cultures of Pseudokirchneriella subcapitata were exposed to Rhamnolipids (84% a.i.) at nominal concentrations of 0,9.39, 20.7, 45.5, 100 and 220 mg test item/L (equivalent to 7.89, 17.4, 38.2, 84.0 and 185 mg a.i./L) under static conditions. Evaluation was based on the nominal concentrations, since all measured concentrations of the test item did not deviate more than 20% from the nominal values.

The NOEC, EC10 and EC50 values based on growth rate and yield were >/=185, >185 and <185 mg a.i./L, respectively.  The % growth inhibition in the treated algal culture as compared to the control ranged from -4.73 to 1.39. No morphological abnormalities were noted.

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata

Test Type: Static

 

72 hr ErC10 / EyC10 >185 mg a.i./L

72 hr ErC50 / EyC50 > 185mg a.i./L

72 hr NOEC >/= 185mg a.i./L

Endpoint(s) Effected:  growth rate, yield

Description of key information

a guideline study on the toxicity of the substance to a freshwater alga (Pseudokirchneriella subcapitata) was conducted, and from which the 72 h ErC10 and 72 h ErC50 values were derived as >185 mg a.i./L

Key value for chemical safety assessment

EC50 for freshwater algae:

185 mg/L

EC10 or NOEC for freshwater algae:

185 mg/L