Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2010 - 25 June 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP-guideline study with acceptable restrictions. Restrictions: 1) undissolved test substance was present at the limit concentration after two days of exposure, 2) dissolved oxygen concentrations had dropped to 3.1 mg/L after two days of exposure.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples (40 mL) for TOC-analysis were taken from the test concentration and the control at t=0, 24 and 96 h.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis.

Additionally, reserve samples of 40 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a refrigerator for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of Vinasses tested was a dark brown suspension that was completely soluble in test medium at the concentration tested.
A loading rate of 100 mg/L was magnetically stirred for 15 minutes to accelerate the dissolving of the test substance in the test medium. The final test solution was clear and yellow.
- Controls: Adjusted ISO medium, formulated using RO-water.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758.
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation (length definition, mean, range and SD): Final test: 2.5 ± 0.1 cm
- Weight at study initiation (mean and range, SD): Final test: 0.40 ± 0.08 g
- Feeding in husbandry: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): same as the test
- Health during acclimatisation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

20.8 - 21.2 °C (control)
20.8 - 21.3 °C (100 mg/L)

pH:

7.5 - 8.0 (control)
7.2 - 7.7 (100 mg/L)

Dissolved oxygen:

6.9 - 9.3 mg O2/L (Control)
3.1* - 9.1 mg O2/L (100 mg/L)
* Oxygen concentration increased to 8.6 mg/L after 8 hours of aeration

Nominal and measured concentrations:

Nominal concentrations: Control and 100 mg/L
Measured concentrations: Based on the TOC content of Vinasses (i.e. 25.96%) the concentration of Vinasses in the aquatic samples was calculated and corrected for the control. The initial concentration was below nominal, i.e. 71 mg/L, and this concentration decreased during the test period. Decrease of test substance concentrations may at least partly be explained by the fish being able to consume Vinasses. Based on these results the average exposure concentration was calculated to be 58 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 6.5 litres, all-glass, containing 6 litres of test solution
- Aeration: Aeration was introduced twice during the test period. The first time after 2 days of exposure and it was maintained for 8 hours. The second time after 3 days of exposure until the end of the test.
- No. of organisms per vessel: 7 fish per concentration
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.47 g fish/litre, i.e. 7 fish per 6 litres of test medium
- Feeding during the test: No feeding

TEST MEDIUM / WATER PARAMETERS
Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature, Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Photoperiod: 16 hours light; 8 h dark

EFFECT PARAMETERS MEASURED: Mortality was recorded at 4, 24, 48, 72 and 96 h following the start of exposure. In addition, every afternoon from day 0 and every morning from day 1 any dead or severely distressed fish were observed.

TEST CONCENTRATIONS
- Test concentrations: control and 100 mg/L

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to test material

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to test material

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: No mortality in control
- Abnormal responses: No abnormalities were observed
- Effect concentrations exceeding solubility of substance in test medium: yes, after two days of exposure the limit concentration was observed to be hazy. The following days undissolved test substance was seen in the test vessel. This indicated that the nominal concentration was above the solubility limit of Vinasses in test medium.

Results with reference substance (positive control):

The 96 h-LC50 for carp exposed to PCP was estimated to be 0.15 mg/L (95% confidence interval between 0.10 and 0.22 mg/L). This effect was already reached within 24 hours of exposure.

Sublethal observations / clinical signs:

Under the conditions of the present test Vinasses induced no visible effects in carp at a nominal concentration of 100 mg/L (NOEC), corresponding to an average exposure concentration of 58 mg/L. The initial concentration was below nominal, i.e. 71 mg/l, and this concentration decreased during the test period. Decrease of test substance concentrations may at least partly be explained by the fish being able to consume Vinasses. Undissolved test substance was seen in the test vessel during exposure. This indicated that the nominal concentration was above the solubility limit of Vinasses in test medium and was an additional explanation for the measured decrease of test substance concentration.

Table 1: Measured Total Organic Carbon content of Vinasses

Time of sampling (hours)	Date of sampling	Date of TOC analysis^1	Nominal conc. (mg/L)	Measured organic carbon conc. (mg/L)	Calculated conc. Vinasses (mg/L)
0	21-06-2010	06-07-2010 28-06-2010	0 100	0.1683 18.61	0 71
24	22-06-2010	06-07-2010 28-06-2010	0 100	0.1301 17.34	0 66
96	25-06-2010	06-07-2010 28-06-2010	0 100	0.2981 12.02	0 45

¹Reserve samples were used to measure the control due to an analytical error in the lowest calibration point

Table 2: Incidence of mortality and total mortality during the limit test

Nominal conc. Vinasses (mg/L)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		4 h	24 h	48 h	72 h	96 h
control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present test Vinasses induced no visible effects in carp at a nominal concentration of 100 mg/L (NOEC), corresponding to an average exposure concentration of 58 mg/L.
The 96h-LC50 was above a nominal concentration of 100 mg/L. Based on non-specific analysis the 96h-LC50 was determined to be > 58 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to read-across justification attached in chapter 13.

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

other: Total Organic Carbon (TOC) converted to test material

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

other: Total Organic Carbon (TOC) converted to test material

Basis for effect:

mortality (fish)

Description of key information

LC50(96h) > 100 mg/L (nominal); > 58 mg/L (measured)

Additional information

The short-term toxicity of Vinasses, residue of fermentation towards fish was tested according to the OECD Guideline 203 “Fish, Acute Toxicity Test”. Cyprinus carpio was used for testing in a static limit test (loading 100 mg/L, nominal). After a test duration of 96 h, no mortality or other abnormal responses were observed. Based on the TOC content of Vinasses (i.e. 25.96%) the concentration of Vinasses in the aquatic samples was calculated and corrected for the control. Thus, the test resulted in an LC50 (96 h) of > 58 mg/L based on the average exposure concentration.

During the test, dissolved oxygen concentration had dropped to 3.1 mg/L in the limit concentration (after 2 days of exposure). Therefore, aeration was introduced immediately. No fish mortality or any other clinical effects were observed. Furthermore, after two days of exposure the limit concentration was observed to be hazy. The following days undissolved test substance was seen in the test vessel. The study author concluded that the applied concentration was above the limit of water solubility of Vinasses, residue of fermentation. There is no test on water solubility of this test substance available. Whereas, for the similar UVCB substance, Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum, it was shown that a large fraction of components with relatively high water solubility is existent (OECD 105). However, the test substance was not completely soluble in water since in all samples undissolved test substance particles were observed. Nevertheless, large fractions exhibited a very high water solubility (38g/L at a loading rate if 100g/L, pH 3.5-3.6). Therefore, it can be concluded, that the observed hazyness and partly precipitations are rather a result of the variable composition of this test substance, than of a limited water solubility. It is likely that both bacterial activities together with the variable composition of the substance resulted in the observed oxygen depletion and precipitation.