Willow, Salix alba, ext.

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:

Experimental phase: 2019-02-22

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 (Carbon and Ammonium Oxidation))

Version / remarks:

adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

according to the most recent amendments of COMMISSION REGULATION (EC) No 440/2008 by Commission Regulations (EU) 2016/266 of 7 December 2015 and 2017/735 of 14 February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Version / remarks:

Product Properties Test Guideline OCSPP 850.3300 (Public Draft OPPTS 850.6800, April 1996) of the United States Environment Protection Agency (EPA) "Modified Activated Sludge, Respiration Inhibition Test", June 2012

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

See IUCLID section "Any other information on materials and methods incl. tables" below.

Vehicle:

no

Details on test solutions:

Just before the start of the experiment a stock solution / dispersion (3200 mg/L) was prepared by dissolving / dispersing powdered test item in deionised water. This stock solution was continuously stirred until homogeneity. The test solutions used in the test were freshly prepared by dilution of this stock solution with deionised water at the beginning of the experiment.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge, microorganisms from a domestic waste water treatment plant.
The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning:
The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. The sludge was fed daily with 50 mL synthetic sewage (according to OECD 209) per litre and kept aerated at 20 +/- 2°C until use.
The activated sludge was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight was determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.31. The activated sludge was used directly thereafter.

A non-GLP internal complementary experiment was performed to demonstrate if any significant change in the activity of the sludge occurred upon cultivation for 48 hours under the same laboratory conditions as in this study. After evaluation of the data, it was concluded that after 48 hours incubation of sludge, there was no significant reduction in nitrification respiration rate: no significant declining of nitrification activity was detected upon storage.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

not relevant

Test temperature:

20.3 – 21.8 °C

pH:

Test item treatments biotic (10, 100 and 1000 mg/L):
start: 7.48 to 7.56; end: 8.10 to 8.18;

Test item treatments abiotic (1000 mg/L):
start: 7.51 to 7.59; end: 7.60 to 7.62;

Inoculum blanks:
start: 7.55 to 7.69; end 7.59 to 8.21;

Reference item treatments (1, 3.2, 10, 21, 32 mg/L) biotic:
start: 7.59 to 7.60; end: 8.00 to 8.05;

Dissolved oxygen:

Saturation level during the test (aeration); measurement endpoint is oxygen depletion (consumption rate).

Salinity:

not applicable

Conductivity:

not applicable

Nominal and measured concentrations:

Nominal test item concentrations:
The test was performed as a combined range finding and limit test. The following test item concentrations were applied:
10 mg/L; 100 mg/L; 1000 mg/L (limit concentration).

Details on test conditions:

See IUCLID section "Any other information on materials and methods incl. tables" below.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: no inhibition of total respiration observed at the limit concentration, rather slight stimulation by 1.12% relative to control

Details on results:

See IUCLID section "Any other information on results incl. tables" below.

Results with reference substance (positive control):

See IUCLID section "Any other information on results incl. tables" below.

Reported statistics and error estimates:

Determination of oxygen uptake inhibition by 50% (EC50)
The 3-hour EC50 value of the reference item and its 95%-confidence limits were calculated by Probit analysis using TOXSTAT software. For the test item the EC50 value was determined directly from the values of percentage of inhibition (no relevant effects observed).

Details on results: 

Control respiration:

The average specific respiration rate (R_S) of the blank controls (B1-B6) was 20.17 mg O₂/(g*h), whereof the oxygen uptake rate of the blank controls B2, B4 and B5 were but slightly below 20 mg O₂/(g*h) (19.31; 19.67; 19.87). This is within the historical control data range and does not affect the validity of the study.

 The variation coefficient of oxygen uptake rate in the blank controls was 3.48%.

 

Respiration inhibition by the test item:

The test item (10–1000 mg/L) inhibited the respiration rate (total oxygen consumption (IT)) of the activated sludge by -1.12% to 6.91%. Inhibition extent was not related to test iem concentration.

Details on meaurements:

Test group	Conc. of test item in test mix.	No of replicates	Total oxygen consumption rate(RT)	Specific respiration rate(RS)	RSD	Inhibition	pH start	pH end
Name, ID	mg/L	TI1 to TI3	mg O2/(L*h)	mg O2/(g*h)	[%]	%
A1	1000	---	0	---		---	7.59	7.62
A2	1000	---	-0.08	---		---	7.51	7.6
A3	1000	---	-0.17	---		---	7.58	7.6
A4	1000	---	-0.17	---		---	7.57	7.6
A5	1000	---	-0.33	---		---	7.57	7.6
B1	0	---	31.85	21.24		---	7.69	7.59
B2	0	---	29.8	19.87*		---	7.59	8.04
B3	0	---	30.4	20.27		---	7.55	8.16
B4	0	---	29.51	19.67*		---	7.59	8.19
B5	0	---	28.97	19.31*		---	7.66	8.18
B6	0	---	31	20.67		---	7.64	8.21
REF1	1	---	23.89	15.92		20.56	7.6	8
REF2	3.2	---	19.02	12.68		36.64	7.6	8.01
REF3	10	---	16.42	10.94		45.25	7.6	8.02
REF4	21	---	9.72	6.48		67.38	7.6	8.04
REF5	32	---	6.08	4.05		79.41	7.59	8.05
TI1	10	5	29.66	19.78	2.6%	1.46	7.55	8.15
TI2	100	5	28.02	18.68	3.7%	6.91	7.56	8.18
TI3	1000	5	30.45	20.3	4.2%	-1.12	7.48	8.1

A1 -5: abiotic control replicates; B1 -6: inoculum blank controls; REF1 -5: concentrations of the reference substance 3,5-dichlorophenol; TI1 -3: concentration levels of the test item;

Validity criteria fulfilled:

yes

Conclusions:

The following results were obtained from a valid respiration inhibition test (OECD 209; compliant with GLP):
EC50 (3 h; total respiration) > 1000 mg/L test item;
NOEC (3h; total respiration) ≥ 1000 mg/L test item.

Executive summary:

In a valid activated sludge respiration inhibition test (combined range-finding and limit test) performed compliant with GLP according to OECD 209 (adopted 2010), no relevant inhibiting effects of the test item could be determined:

Inhibition of total respiration at 10 mg/L test item amounted to 1.46% of control respiration;

Inhibition of total respiration at 100 mg/L test item amounted to 6.91% of control respiration;

Inhibition of total respiration at 1000 mg/L test item amounted to minus 1.12% of control respiration, i.e. respiration was slightly increased compared to the control.

In conclusion, at all tested concentrations inhibition of total respiration was below 10% of control respiration. Because an EC10 value may be used as a substitute for a NOEC value, and no dose-response relationship becomes obvious from these data, the observed effects at 10 and 100 mg/L may be regarded as biologically not relevant and the tested limit concentration of 1000 mg/L test item is regarded as the no-effect concentration (NOEC):

EC50 (3 h; total respiration) > 1000 mg/L test item;

NOEC (3h; total respiration) ≥ 1000 mg/L test item.

In consequence, according to OECD 209 (paragraph 40) no further testing was necessary: the test item was determined to be not inhibitory to activated sluge respiration.

Description of key information

The following results were obtained from a valid respiration inhibition test (OECD 209; compliant with GLP):

EC50 (3h; total respiration) > 1000 mg/L test item;

NOEC (3h; total respiration) ≥ 1000 mg/L test item.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In a valid activated sludge respiration inhibition test (combined range-finding and limit test) performed compliant with GLP according to OECD 209 (adopted 2010), no relevant inhibiting effects of the test item could be determined:

Inhibition of total respiration at 10 mg/L test item amounted to 1.46% of control respiration;

Inhibition of total respiration at 100 mg/L test item amounted to 6.91% of control respiration;

Inhibition of total respiration at 1000 mg/L test item amounted to minus 1.12% of control respiration, i.e. respiration was slightly increased compared to the control.

In conclusion, at all tested concentrations inhibition of total respiration was below 10% of control respiration. Because an EC10 value may be used as a substitute for a NOEC value, and no dose-response relationship becomes obvious from these data, the observed effects at 10 and 100 mg/L may be regarded as biologically not relevant and the tested limit concentration of 1000 mg/L test item is regarded as the no-effect concentration (NOEC):

EC50 (3 h; total respiration) > 1000 mg/L test item;

NOEC (3h; total respiration) ≥ 1000 mg/L test item.

In consequence, according to OECD 209 (paragraph 40) no further testing was necessary: the test item was determined to be not inhibitory to activated sluge respiration.