fermentation residues of molasses, yeast extract, sucrose and ammonia with bacillus subtilis

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 October 2009 - 20 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Biomass residue (liquid) obtained after B2 production

IUPAC Name:

Biomass residue (liquid) obtained after B2 production

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for Total Organic carbon (TOC) analysis were taken from the highest test concentration and the control according to the schedule below.

Frequency: at t=0 h and t=48 h
Volume: 40 ml
Storage: Samples were stored in a refrigerator until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Biomass residue (liquid) obtained after B2 production tested was a brownish-yellow turbid liquid containing 25-35% dry matter in water. Although the test substance was indicated to be soluble in water, it was not completely soluble in ISO-medium at loading rates of 10 and 100 mg/l (based on the liquid suspension as received). This is probably due to the salts in the ISO-medium.

Preparation of the test solutions started with loading rates of 1.0, 10 and 100 mg/l applying 1 day of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures, of which the 1.0 mg/l loading rate was observed to be clear and colourless while the higher loading rates were turbid and increasingly yellow, were filtered through a glass fibre pre-filter (Whatman). The Water Soluble Fractions (WSFs) were used for testing together with a 10% dilution prepared from the 1.0 mg/l WSF.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age at study initiation: < 24 hours
- Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 liters of medium in an all-glass culture vessel.
- Feeding during test: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

Between 19.3 and 20.0°C

pH:

Between 7.5 and 7.8

Dissolved oxygen:

Between 6.8 and 9.1 mg/l

Nominal and measured concentrations:

Nominal concentration: 0 (control), 10% of a WSF prepared at a loading rate of 1.0 mg/l and WSFs prepared at loading rates of 1.0, 10 and 100 mg/l (concentrations based on the liquid suspension as received).
Measured concentartions: 0 - WSF prepared at a loading rate of 100 mg/l
Based on the TOC content of Biomass residue (liquid) obtained after B2 production (i.e. 13%) the concentration of Biomass residue (liquid) obtained after B2 production in the aquatic samples was calculated and corrected for the control. The initial concentration in the WSF prepared at a loading rate of 100 mg/l was calculated to be 38 mg/l. This concentration decreased to 20 mg/l at the end of the test. Based on these results the average exposure concentration was calculated to be 27 mg/l.

Details on test conditions:

- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 100 ml, normal headspace
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest concentration, 2 for the lower test concentrations
- No. of vessels per control (replicates): 4
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts:
CaCl2.2H2O : 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO3 : 46.7 mg/l
KCl : 4.2 mg/l
The hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.
- Culture medium different from test medium: yes, for culturing M7 medium is used
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC50 t=48 h
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: Control, 10% of a WSF prepared at a loading rate of 1.0 mg/l and WSFs prepared at loading rates of 1.0, 10 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon converted to test material

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to test material

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: mg/l WSF prepared at 100 mg/l)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg/l (WSF prepared at 100 mg/l)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Behavioural abnormalities: no
- Immobility of control: 5% immobility was observed.
- Other adverse effects control: no

Results with reference substance (positive control):

The 24h-EC50 was 0.84 mg/l with a 95% confidence interval between 0.78 and 0.94 mg/l.
The 48h-EC50 was 0.75 mg/l with a 95% confidence interval between 0.56 and 1.0 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Biomass residue (liquid) obtained after B2 production did not induce acute immobilisation of Daphnia magna at a loading rate of 100 mg/l (NOEC), corresponding to an average exposure concentration of 27 mg/l.
The 48h-EC50 was above an initial loading rate of 100 mg/l. Based on non-specific analysis the 48h-EC50 was determined to be >27 mg/l.