Vinasses, residue of fermentation

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:

21 June 2010 - 24 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 40 mL samples for TOC-analysis were taken from the test concentration and the control at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis.

At the end of the exposure period, the replicates without algae were pooled at each concentration before sampling.
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 17 minutes to accelerate the dissolving of the test substance in the test medium. The final test solution was clear and yellow-brown.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 1.0 * 10E4 cells/mL.
- Controls: Test medium without test substance or other additives

Test organisms (species):

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

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/mL.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m²/s) when measured in the photosynthetically effective wavelength range of 400 to 700 nm in a climate room at a temperature of 21-24 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol (24 mg CaCO3/L)

Test temperature:

22.7 - 23.3 °C

pH:

7.9 - 8.2 (control)
7.8 - 7.9 (100 mg/L)

Salinity:

not applicable

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. 75 mg/L, and this concentration decreased during the test period. Based on these results the average exposure concentration was calculated to be 56 mg/L.

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: all-glass, 100 mL, normal headspace, 50 mL
- Aeration: no
- Initial cells density: 10000 cells/mL
- Control end cells density: 965000 cells/mL
Replicates:
6 replicates of both the control and the limit concentration;
4 extra replicates of both the control and the limit concentration without algae for sampling purposes;
1 extra replicate of the highest concentration without algae as background for the determination of the algal cell density.

GROWTH MEDIUM
- Standard medium used: yes / M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 85 to 92 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
spectrophotometric measurement of cell densities, daily

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

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to test material

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) convereted to test material

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) convereted to test material

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) convereted to test material

Basis for effect:

other: Yield

Details on results:

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Effect concentration exceeding solubility of substance in te test medium: Yes, from two days of exposure onwards the limit concentration was observed to contain undissolved particles. This indicated that the nominal concentration was above the solubility limit of Vinasses in test medium and was an explanation for the measured decrease of test substance concentration.

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72 h) was 1.1 mg/L with a 95% confidence interval ranging from 1.0 to 1.3 mg/L.
The EC50 for yield inhibition (EYC50: 0-72 h) was 0.59 mg/L with a 95% confidence interval ranging from 0.51 to 0.68 mg/L.

Reported statistics and error estimates:

For determination of the NOEC the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Tukey test, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). No EC50-values could be calculated because the test substance did not cause ≥50% effect.

Under the conditions of the present study Vinasses did not induce growth rate reduction at a nominal concentration of 100 mg/L (NOEC), corresponding to an average exposure concentration of 56 mg/L. Vinasses induced 23% inhibition of yield at a nominal concentration of 100 mg/L, i.e. the NOEC was below a loading rate of 100 mg/L corresponding to an average exposure concentration of 56 mg/L. The 72 h-EC₅₀ for both growth rate reduction and yield inhibition was above a nominal concentration of 100 mg/L. Based on non-specific analysis the 72 h-EC₅₀ was determined to be > 56 mg/L.

Table 1: Calculation of growth rate (section-by-section)

Nominal conc.	Vessel	Growth rate (µ)			Growth rate reduction (%)
Vinasses	number
(mg/L)		0-24 h	24-48 h	48-72 h	0-24 h	24-48 h	48-72 h
control	1	0.06501	0.06236	0.06639
	2	0.06579	0.05930	0.06159
	3	0.06276	0.06179	0.06605
	4	0.06483	0.06242	0.06674
	5	0.06457	0.05983	0.06182
	6	0.06457	0.06156	0.06436
	mean	0.06459	0.06121	0.06449
	CV	2%	2%	4%
	The mean CV for section-by-section specific growth rate was:				4%
100	1	0.08351	0.03479	0.06015	-29	43	7
	2	0.08937	0.02647	0.06432	-38	57	0
	3	0.08440	0.03079	0.06167	-31	50	4
	4	0.08612	0.03843	0.05680	-33	37	12
	5	0.08516	0.03284	0.06172	-32	46	4
	6	0.08664	0.03199	0.06414	-34	48	1
Mean					-32.9	46.8	4.7

Table 2: Measured Total Organic Carbon content

Time of sampling (hours)	Date of sampling	Date of TOC analysis	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	0.2667 19.83	0 75
24	22-06-2010	06-07-2010 28-06-2010	0 100	0.3060 14.89	0 56
72	24-06-2010	06-07-2010 28-06-2010	0 100	0.4448 12.59	0 47

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Vinasses did not induce growth rate reduction at a nominal concentration of 100 mg/L (NOEC), corresponding to an average exposure concentration of 56 mg/L.

Vinasses induced 23% inhibition of yield at a nominal concentration of 100 mg/l, i.e. the NOEC was below a loading rate of 100 mg/l corresponding to an average exposure concentration of 56 mg/l.

The 72h-EC50 for both growth rate reduction and yield inhibition was above a nominal concentration of 100 mg/l. Based on non-specific analysis the 72h-EC50 was determined to be >56 mg/l.

Description of key information

EC50(72h) > 100 mg/L (nominal), EC50 > 56 mg/L (measured) - for growth

NOEC(72h) = 100 mg/L (nominal), NOEC = 56 mg/L (measured) - for growth

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

56 mg/L

Additional information

The toxicity of Vinasses, residue of fermentation towards aquatic algae was investigated in a study conducted according to the OECD Guideline 201 “Alga, Growth Inhibition Test”. The freshwater algae Pseudokirchneriella subcapitata was used as test organism and exposed to a nominal concentration of 100 mg/L for 72 h. Based on the TOC content of the test material (i.e. 25.96%) the concentration of Vinasses in the aquatic samples was calculated and corrected for the control. No acute effects were observed based on growth rate and yield (EC50(72h) > 56 mg/L, measured, EC50(72 h): > 100 mg/L, nominal). Furthermore, under the conditions of the present study Vinasses did not induce growth rate reduction at a nominal concentration of 100 mg/l (NOEC), corresponding to an average exposure concentration of 56 mg/L. However, the test substance induced 23% inhibition of yield at a loading rate of 100 mg/L, i.e. the NOEC was below a loading rate of 100 mg/L corresponding to an average exposure concentration of 56 mg/L (for yield).

 

Similar to observations in the acute fish test on this substance, after two days of exposure onwards the limit concentration was observed to contain undissolved particles. There is no test on the water solubility of this test substance available, but 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 of 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 precipitation.