Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 10, 2019 - July 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

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 No. FD 1851T1019
- Purity, including information on contaminants, isomers, etc.: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Recommend to store the sample dry and below 30°C.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in container after opening. Soluble in water, stable

Analytical monitoring:

yes

Details on sampling:

Liquid Chromatography Mass Spectrometer (LC-MS/MS)

Vehicle:

yes

Remarks:

Dechlorinated tap water

Details on test solutions:

0.050 g, 0.089 g, 0.158 g, 0.281 g and 0.500 g test substance were weighed into flasks respectively and dissolved with some amount of test water. After ultrasoni treatement for 15 min, the solution were respectiveley transferred into 5 L vessels and diluted with test water to 500 mL to obtain the nominal concentrations of 19.0 mg/L, 17.8 mg/L, 31.6 mg/L, 56.2 mg/L and 100 mg/L test solutions, which were used direclty in the definitive test.

Test organisms (species):

other: Rare Minnow (Gobiocypris rarus)

Details on test organisms:

TEST ORGANISM
- Common name: Rare Minnow
- Strain: Gobiocypris rarus
- Source: test laboratory

ACCLIMATION
- Acclimation period: 112 days
- Acclimation conditions: same as test
- Feeding frequency during acclimation:same as test tim every workday until 24 hpurs before the start of the test,
- Health during acclimation (any mortality observed): The mortality was 0% during the 7 days prio the start of the test, which met the requirement of mortalities being less than 5% of the population

Test type:

semi-static

Water media type:

freshwater

Remarks:

Dechlorinated tap water

Total exposure duration:

96 h

Hardness:

120 mg/L expressed as CaCO3

Test temperature:

23.2 - 23.7 °C

pH:

8.29

Dissolved oxygen:

The dissolved oxygen in the test solutions during the test was kept above 60% of the air saturation value.

Nominal and measured concentrations:

Nominal Concentrations: 10.0 mg/L, 17.8 mg/L, 31.6 mg/L, 56.2 mg/L and 100 mg/L test solutions-
Measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 5L / test vessel
- Material, size, headspace, fill volume:
- Aeration: no areation
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 7 fish /group , 42 fish in total
- No. of vessels per concentration (replicates): No replicates
- No. of vessels per control (replicates): No replicates
- No. of vessels per vehicle control (replicates): No replicates
- Loading of test fish: 7 fish / test vessel, 0.169 g fish / L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 14 h light / 10 h darkness
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
1. Measurements of the light illumination; the intensitiy of illumination was measured at the beginning and end of the test.
2. Mortalities and visible abnormalities of the test fish were recorded at 3 h, 24 h, 48 h, 72 h and 96 h after the start of the test during test peiod. the dead fish were removed as soon as the were found. A fish was considered deadm if there was no visible movement (e.g gill movements) and if touching of the caudal dpeduncle procedures no rection.
3. Observation of test solutions appearance: The changes of cloro and turbidity of the test solutions were recorded daily.
4. Measure,ent of temperature, pH, and dissolved oxygen of test solution: The temperature, pH and dissolved oxygen of each test groups were measured at the start and the end of test and measured before and after each renewal. Measurements of pH were measured with a portable pH meter. Measurements of temperature and dissolved oxygen were measured with an oxygen meter. The temperature of the test water was continuosly monitored during the test expsoure by a termometer.
5) Body weight and and total lenght of test fish
The body weight and total lenght of 7 test fish in the blank control were measured at the end of the test.
6) Cncentration analysis of the test substance in test solution
The measured concentrations of the test substance in each test groups were measured at 0 h, 72, h (new solution) and 24 h, 96 H (old) solution) by LC-MS/MS, and the blank control was measured at 0 h and 24 h (old solution)

Reference substance (positive control):

yes

Remarks:

K3Cr207

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

562 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Preliminary tests (non-GLP): In the range-finding test, the mortalities in the blank control and 10 mL, 30 mL and 100 mg/L test groups were 0%, 0%, 0% and 100% respectively. Hte analytical result of range-finding test showed that, the measured concentration of test substnace in test solution of 30 mL and 100 mg/L were maintained within 80% - 120% of the nomimal concentration for 24 h (See Attachment unr Overal remarks)

Intensity of illumination: At the start of the test, the intensitties of illumination of the test environment were: 613 lx, 764 lx, 619lx (665 lx on average). And end of the test, the intensities of illumination of the test environment were: 696 lx, 780 lx, 597 lx (691 lx on average).

Observation of test solutions: Test solution of the blank control and all test groups were pobserved to be clear and colorless during the test period.

The acute toxicity of the test substance to Rare Minnow (Gobiocypris rarus) was performed as definitive test under semi-static conditions (24h intervals). At the end of the test, the cumulative mortalities of the nominal concentration of 10.0 mg/L, 17.8 mg/L, 31.6 mg/L, 56.2 mg/L and 100 mg/L test groups were 0%, 0%, 0%, 0% and 100%, respecively. Thus, the effects of the test substance to Rare Minnow described as the 96 h median lethal concentration (LC50), the lowest concentration causing 100% mortality (LC100), and the highest concentration causing 0% mortality (LC50) are respectively: 75.0 mg/L. 95% (confident limit could not be estimated)), 100 mg/L and 56.2 mg/L.

Observed abnormal response: the abnormal responses observations during the test are shown in the attachement under overall results. No abnormal responses of the test fish were observed in the blanck control and 10.0 mg/L, 17.8 mg/L, 31.6 mg/L, and 56.2 mg7L test groups. Reduction of activity was observed in the test fisgh of 100 mg/L test group, all test sih were died at 24 h after start of the test
Body weight and total lenght of test fish: The average and standard deviation of body weight and lenght of the test fish are shown in the attachment in Overall remarks. The aveerage and standard deviation of body weight and total lenght of 7 test fish were 0.121 g +- 0.021 g and 25.51 mm+- 1.51 mm, respectively. So loading rate of fish in the test was 0.169 g/ fish / L , which met the require,ent of <= 1.0 g fish / L and total length was within the range of 30 mm+- 10 mm.

Reported statistics and error estimates:

Cumulative mortalities of each test group at 24h, 48h, 72h and 96h after the start of the test were calculated. the LC50 values of test substance to Rare Minnow at 24h, 48h, 72h and 96h were calculated by Trimmed Spearman Karber Method (US EPA data stat).

Validity criteria fulfilled:

yes

Remarks:

- The mortality of blank control was 0% at the end of the test (the mortality should not exceed 10%) - The DOC in the test solutions ranged from 70% to 96% of the air saturation value throught the test - The test met all control requirements

Conclusions:

The acute toxicity of test substance to Rare Minnow was performed as definitive test under semi-static conditons (24h Intervals). At the end of the test, the cumulative mortalities of the nominal concentration of 10.0 mg/L , 17.8 mg/L, 31. 8 mg/L, 56.2 mg/L and 100 mg/L test groups were 0%, 0%, 0% and 100%, respectively. Hence the effects of the test substance to Rare Minnow described as the 96 h median lethaly concentration (LC50), the lowest concentration causing 100% mortality (LC100) and the highest concentration causing 0% mortality are: LC50 75 mg/mL, LC 100 100 mg/L and LC0 56.2 mg/L.

Description of key information

An acute toxicity study (OECD 203) test on the effects of Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria on Rare Minnow was conducted. At the end of the test, the cumulative mortalities of the nominal concentration of 10.0 mg/L, 17.8 mg/L, 31. 8 mg/L, 56.2 mg/L and 100 mg/L test groups were 0%, 0%, 0% and 100%, respectively. Hence the effects of the test substance to Rare Minnow described as the 96 h median lethal concentration (LC50), the lowest concentration causing 100% mortality (LC100) and the highest concentration causing 0% mortality are: LC50 75 mg/mL, LC100 mg/L and LC0 56.2 mg/L. Thus, the LC50 (96h) is Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria 75 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

ca. 75 mg/L