Vinasses, residue of fermentation, salt-enriched

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 0.5 g/L was prepared by adding 0.1252 g test substance to 250 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (15 minutes) was used to accelerate dissolution and to ensure homogeneity. Volumes of the clear brown stock solution corresponding to the test concentration were then added to the test media. A concentration of 100 mg/L was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge was obtained from the municipal sewage treatment plant "Watershap Aa en Maas', 's-Hertogenbosch, The Netherlands. The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3.8 g/L of sludge, as used for the test). The pH was 8.0 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours exposure period.

Test temperature:

18.3 - 18.7 °C

pH:

8.1 - 8.3

Dissolved oxygen:

6.7 - 8.1 mg O2/L

Salinity:

not applicable

Nominal and measured concentrations:

nominal concentration 100 mg/L

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 mL oxygen bottles (measuring) and 1 litre test bottles (exposure)
- Aeration: During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2/series
- Biomass loading rate: 1.5 g/L suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q).

EFFECT PARAMETERS MEASURED: The respiration rate from each vessel, in mg O2/L/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/L.

TEST CONCENTRATIONS
- Test concentrations: limit study at 100 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

No (significant) inhibition of respiration rate of the sludge was recorded at 100 mg Vinasses per litre. The duplicate measurement confirmed the result of the first measurement. Hence, the EC50 of Vinasses exceeded 100 mg/L (based on nominal concentrations).

Results with reference substance (positive control):

Results with reference substance valid: Yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing concentration, ranging from a 35 % inhibition at 5.0 mg/L to 75% at 30 mg/L. Relevant effects levels: The EC50 of 3,5-dichlorophenol was 9.3 mg/L.

Reported statistics and error estimates:

For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For Vinasses no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 100 mg/L).

Under the conditions of this present test, Vinasses was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/L. The EC₅₀ of Vinasses exceeded 100 mg/L (based on nominal concentrations).

Table 1: Controls (C) and Vinasses (T): pH, oxygen concentration, oxygen consumption and percentage inhibition of the respiration rate

Flask	Concentration (mg/L)	Oxygen conc. at the start (mg O2/L)	Oxygen consumption (mg O2/L/h)	% Inhibition respiration rate	pH
Cstart T	0	7.3	44	-	8.2
Cend T	0	8.2	39	-	8.3
			∆ 13%
T 1	100	6.7	47	-13¹	8.1
T 2	100	8.1	41	1	8.3

¹: A negative value indicates stimulation of the sludge. This value is considered as not significant.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test, Vinasses was not toxic to waste water (activated sludge) bacteria at a nominal concentration of 100 mg/L. The EC50 of Vinasses exceeded 100 mg/L (based on nominal concentrations).

Reference 2

Endpoint:

activated sludge respiration inhibition testing

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:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Description of key information

EC50 (3h) > 100 mg/L

Additional information

One study investigating the effects of Vinasses, residue of fermentation to microorganisms is available. This study was performed according to the OECD guideline 209 “Activated Sludge, Respiration Inhibition Test” using activated sludge of a predominantly domestic sewage. No inhibition of the respiration rate was observed, resulting in an EC50 (3 h) of > 100 mg/L (based on nominal concentrations). The observation of no toxic properties of the test substance Vinasses, residue of fermentation is underlined by the absence of inhibiting effects in the toxicity control in an OECD 301B test (see technical dossier chapter 5.2.1). Therefore, a disturbance in the biodegradation process of sewage treatment plant is not anticipated.