Fast Pyrolysis Bio-oil

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 2013 – 19 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

2010

Deviations:

yes

Remarks:

due to practicability the number of replicates for the main test were reduced; the temperature was above 20°C ±2°C

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The main test was performed with the following concentrations: 9.54, 30.5, 97.7, 312.5 and 1000 mg/L. The test item amounts were weighed directly onto a Teflon tape and given into the test vessels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant of Pforzheim, Germany, collected from the aeration tank, was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage.
The sludge was used one day after collection. It was settled for about 10 minutes and the upper layer with finer solids was decanted. Before starting the test, it was washed three times with chlorine free tap water by centrifugation (10 minutes at 3000 rpm). After centrifuging, the supernatant was decanted and discarded and the sludge was resuspended in chlorine free tap water.
The mixed liquor suspended solids (MLSS) were adjusted to a concentration of 3.0 g/L (± 10 %). The activated sludge was continuously aerated at the test temperature, the solids did not settle down.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

none

Hardness:

no data

Test temperature:

21.1 – 22.7 °C

pH:

7.00 - 7.41

Salinity:

no data

Nominal and measured concentrations:

The main test was performed with the following concentrations: 9.54, 30.5, 97.7, 312.5 and 1000 mg/L. All test item concentrations were performed with three replicates whereas the control was performed with six replicates. The test item amount were weighed directly onto a Teflon tape and given into the test vessels.

Details on test conditions:

1 litre glass beakers with test volume of 500 mL.; Measurements were taken 3 hours after test start (t0) which was defined as the initial contact of the activated sludge inoculum with the other constituents of the final mixture. The test solution was filled into a BOD-flask and stirred, and the oxygen concentration was measured and recorded over a period of about 5 minutes. All treatments were measured in the same way at approximately 5-minute intervals.
Aeration was performed during the whole test period but not during the measurements.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol (DCP)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total oxygen uptake

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: heterotrophic oxygen uptake

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

246 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Remarks on result:

other: 77.3 - 930

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

27.1

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Remarks on result:

other: 0.343 - 83.5

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

6.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake due to nitrification

Remarks on result:

other: 0.00521 - 30.9

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

97.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total oxygen uptake

Key result

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

312.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total oxygen uptake

Details on results:

The test item Fast Pyrolysis Bio-oil had statistically significant inhibitory effect for the total oxygen uptake at 312.5 and 1000 mg/L. Statistically significant effects for the oxygen uptake due to nitrification were determined for all test item concentrations after 3 hours. No statistically inhibitory effects were determined for heterotrophic microorganism.
The NOEC was determined to be at 97.7 mg/L for the total oxygen uptake. The LOEC was determined to be at 312.5 mg/L for the total oxygen uptake. The lowest test item concentrations (9.54 and 30.5 mg/L) were excluded since they were without the dose-response relation. No NOEC was determined for the oxygen uptake due to nitrification.
The EC50 for the oxygen uptake due to nitrification was determined to be 246 mg/L. No EC50 were calculated for the total and heterotrophic oxygen uptake, because the inhibition was below 50 % at the highest test item concentration.

Results with reference substance (positive control):

The EC50 of the total oxygen uptake for DCP was estimated within a range of 5 to 25 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L. For the heterotrophic oxygen uptake for DCP, the EC50 was determined to be 15.9 mg/L after 3 hours, excluding the reference item concentration of 5 mg/L, which was without the dose-response relation. The recommended range of 5 to 40 mg/L was therefore fulfilled. For the oxygen uptake due to nitrification the EC50 was estimated within a range of 0.2 and 1 mg/L after 3 hours. This was also within the recommended range of 0.1 to 10 mg/L.

Reported statistics and error estimates:

The statistical evaluation was performed for specific respiration rate of the control and the test item concentrations using SAS® (2002–2008). The calculation was performed using SAS Software service pack 9.2. A test for normality of the data was performed by calculating the Shapiro-Wilk’s statistic. A test for homogeneity of variance for the data was performed using the Levene-test. The NOEC was determined using Dunnetts-t- test for the total and for heterotrophic microorganisms (left sided). The NOEC was evaluated using the Jonckheere-Terpstra test for the oxygen uptake due to nitrification. The values were transformed with boxcox transformation, outliers were determined and excluded from the calculation. P-values below 0.05 showed statistically significant differences to the control. The EC50 estimation of the main test was based on the percentage of inhibition, presented in Table 6. The EC50 values for the test item were calculated using the probit analysis following the Gompertz distribution for oxygen uptake due to nitrification. No statistical evaluation was performed for the total and heterotrophic oxygen uptake because the inhibition was below 50% at the highest test item concentration.
The EC50-value for DCP was calculated using Spearman-Kärber estimator (Anellis & Werkowsky, 1968). Due to statistical reasons the inhibition values of 1.0 and 25 mg/L were rounded and included in the calculation.

Table 5:        Results of the main test,specific respiration rates

Test assay	Specific respiration rates [mg O2/(g × h)]
	Total	Heterotrophic	Due to nitrification
nominal [mg/L]	3 h	3 h	3 h
Control (1)	34.23	16.71	11.92^1)
Control (2)	27.43	18.07
Control (3)	26.65	18.31
Control (4)	27.89	16.23
Control (5)	27.67	14.17
Control (6)	28.32	17.19
9.54 (1)	24.96	11.48	9.95^1)
9.54 (2)	24.99	16.77
9.54 (3)	25.33	17.17
30.5 (1)	26.51	17.49	9.45^1)
30.5 (2)	26.47	16.69
30.5 (3)	27.04	17.48

 

Table 5 (continued): Results of the main test, specific respiration rates

Test assay	Specific respiration rates [mg O2/(g × h)]
	Total	Heterotrophic	Due to nitrification
nominal [mg/L]	3 h	3 h	3 h
97.7 (1)	26.87	18.28	7.61^1)
97.7 (2)	24.72	18.85
97.7 (3)	26.77	18.40
312.5 (1)	25.15	17.33	6.86^1)
312.5 (2)	25.49	19.48
312.5 (3)	24.87	17.53
1000 (1)	19.30	17.67	1.97^1)
1000 (2)	19.39	16.80
1000 (3)	20.08	18.39
DCP 0.2	27.23	17.95	9.28
DCP 1	20.47	17.09	3.38
DCP 5	18.50	3.60^2)	14.90^2)
DCP 25	4.41	3.67	0.74

¹⁾these values are the difference between the mean total and the mean of the heterotrophic rates²⁾This reference item concentration with ATU was excluded from the calculation since it is shown on the base of nitrification that this concentration was without the dose response relation.

Table6:        Results of the main test, inhibition

Test assay	Mean inhibition [%]^1)
	Total	Heterotrophic	Due to nitrification
nominal [mg/L]	3 h	3 h	3 h
9.54	12.6*	9.8	16.5*
30.5	7.1*	-2.6	20.7*
97.7	9.0	-10.3	36.2*
312.5	12.3*	-7.9	40.8*
1000	31.7*	-5.0	83.5*
DCP 0.2	5.1	-7.0	22.1
DCP 1	28.7	-1.8	71.6
DCP 5	35.5	78.5^2)	-25.0^2)
DCP 25	84.6	78.1	93.8

¹⁾   negative inhibition values indicate stimulating effects

*   statistically significant different to the control

²⁾   This reference item concentration with ATU was excluded from the calculation since it is shown on the base of nitrification that this concentration was without the dose response relation.

Table7:   Endpoints forFast Pyrolysis Bio-oil

Endpoint	Fast Pyrolysis Bio-oil [mg/L]
	nominal	95% confidential limits
Total EC50	> 1000	-
Heterotrophic EC50	> 1000	-
Due to nitrification EC50^1)	246	77.3 - 930
Due to nitrification EC20^1)	27.1	0.343 – 83.5
Due to nitrification EC10^1)	6.29	0.00521 – 30.9
NOEC (total oxygen uptake)	97.7
LOEC (total oxygen uptake)	312.5

¹⁾Probit analysis following the Gompertz distribution        n.d.: not determined

Validity criteria fulfilled:

yes

Conclusions:

The effect of fast pyrolysis bio-oil on activated sludge microorganisms respiration was tested. The test item had statistically significant inhibitory effect for the total oxygen uptake at loading rates of 312.5 and 1000 mg/L. Statistically significant effects for the oxygen uptake due to nitrification were determined for all test item concentrations after 3 hours. No statistically inhibitory effects were determined for heterotrophic microorganism.

No EC50 were calculated for the total and heterotrophic oxygen uptake, because the inhibition was below 50 % at the highest test item concentration (1000 mg/L). The EC50 for the oxygen uptake due to nitrification was determined to be 246 mg/L. The determined NOEC for total oxygen uptake was 97.7 mg/L (analyzed concentration). No NOEC was determined for the oxygen uptake due to nitrification.

Executive summary:

Toxicity of the fast pyrolysis bio-oil toward microorganisms (activated sewage sludge) was determined in a GLP compliant study according to OECD Guidelines for Testing Chemicals (2010) no 209 "Activated sludge, respiration inhibition test". All validity criteria of OECD Guideline for Testing of Chemicals No. 209 were fulfilled. As a minor deviations from the guideline, due to practicability the number of replicates for the main test were reduced and the test temperature was above 20°C ±2°C.

 

The respiration rate of an activated sludge, fed with a standard amount of synthetic wastewater feed, was measured 3 hours after application of the test item. The inhibitory effect of fast pyrolysis bio-oil in particular concentrations were expressed as a percentage of the mean respiration rate of the controls.

 

The activated sludge used in the test was adjusted to a content of 3.0 g/L dry matter and it was exposed to the test item under continuos aeration. After stopping the aeration, the O₂ consumption was measured for approx. 5 minutes. The slope of the O₂ consumption straight line is an indication for toxic effects on respiration activity of microorganisms.

 

The main test was performed with the following concentrations: 9.54, 30.5, 97.7, 312.5 and 1000 mg/L. All test item concentrations were performed with three replicates whereas the control was performed with six replicates. Four concentrations of 3,5-dichlorophenol were also tested to demonstrate the sensitivity of the test system. To test the differences between total oxygen uptake, heterotrophic oxygen uptake and oxygen uptake due to nitrification, two test assays were performed, one with and one without allylthiourea. The oxygen uptake rate was determined after 3 hours.

 

Statistically significant inhibitory effects for the total oxygen uptake were observed at the two highest loading rates (312.5 and 1000 mg/L). Statistically significant effects for the oxygen uptake due to nitrification were determined for all test item concentrations after 3 hours. No statistically inhibitory effects were determined for heterotrophic microorganism.

 

The determined NOEC for total oxygen uptake was 97.7 mg/L (analyzed concentration). No NOEC was determined for the oxygen uptake due to nitrification.

 

The EC₅₀ for the oxygen uptake due to nitrification was determined to be 246 mg/L. No EC₅₀ values were calculated for the total and heterotrophic oxygen uptake, because the inhibition was below 50 % at the highest test item concentration (1000 mg/L).

The test is reliable without restriction.

Description of key information

Toxicity of the test material toward microorganisms (activated sewage sludge) was determined in a GLP compliant study according to OECD Guidelines for Testing Chemicals (2010) no 209 "Activated sludge, respiration inhibition test". Statistically significant inhibitory effects were observed at the two highest loading rates (312.5 and 1000 mg/L). NOEC for total oxygen uptake was determined at 97.7 mg/L (analyzed concentration). 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

97.7 mg/L

Additional information

Due to poor water solubility of the substance, the organisms were exposed to a Water Accommodated Fraction (WAF) of the test substance. This is a commonly accepted procedure for regulatory studies on poorly water soluble substances.