Ashes (residues), nonhazardous municipal solid waste

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9.11.2007-5.12.2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues)

EC Number:

268-627-4

EC Name:

Ashes (residues)

Cas Number:

68131-74-8

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Main components: SiO2 (42.12%), Al2O3 (32.16%), Fe2O3 (10.88%), TiO2, MnO, MgO, CaO, Na2O, K2O, SO3 - constituents with content less than 10%
- Expiration date of the lot/batch: unlimited
- Appearance: greyish black powder

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION OF SOLUTIONS FOR THE TEST - TEST SUBSTANCE:
The test substance is non-soluble in water in concentrations needed for the test. For this reason the direct dosing of test substance was used. The substance was weighed into reaction container where 100 mL of water were introduced.
In the preliminary test the concentrations were in the range of 46 - 1000 mg.L-1 with a geometric progression factor of 2.2.
In the main test the concentrations were in the range of 100 - 1000 mg.L-1 with a geometric progression factor of 1.8.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

TEST SYSTEM:
The activated biological sludge containing mixed culture of microorganisms obtained from the second step of sewage treatment plant of Pardubice was used for the testing. The wastewater processed by the sewage treatment plant is predominantly municipal.

PREPARATION OF THE INOCULUM:
The activated sludge was collected two days before the day of testing.
After the sample collection the sludge was washed with potable water for 0.5 hour and subsequently decanted for 0.5 hour. This procedure was repeated three times in total.
Further the sludge was modified by addition of 50 mL of cultivation medium per 2 L of diluted sludge suspension at permanent aeration till the day oftest.
The dry weight was determined from 10 mL of sludge suspension after 0.5-hour sedimentation.
Before the test the sludge was suspended in water up to concentration about 4000 mg of sludge dry weight per litre. The pH adjustment to 6.0 was carried out. In that way modified sludge suspension was aerated until the use.

JUSTIFICATION FOR THE SELECTION OF THE TEST SYSTEM:
The activated sludge obtained is in conformity with the recommendations of the test guideline.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

180 min

Test conditions

Test temperature:

20+/-2oC

pH:

approx. 6-8

Nominal and measured concentrations:

Preliminary test: 46, 100, 220, 460, 1000 mg.L-1
Main test: 100, 180, 320, 560, 1000 mg.L-1

Details on test conditions:

TEST PROCEDURE
100 mL of deionised water, the defined dose of the test or reference substance, 16 mL of the synthetic nutrient medium and 200 mL of the prepared sludge suspension were introduced into each test container. The volume was replenished up to 500 mL with deionised water.
The control samples were prepared only from the same volume of nutrient medium and sludge and replenished up to 500 mL with water.
The check of of abiotic decomposition was performed at the highest concentration of the test substance and nutrient medium without sludge and with addition of 1 mL of mercury chloride solution.
The test mixtures were prepared in turns in 15 minutes time intervals.
The pH adjustment of mixtures in the preliminary and in the main test was not carried out.
The aeration was carried out by means of glass pipettes.

MEASUREMENT
The measurement of oxygen content was performed in aliquots of the reaction mixtures after 3-hour incubation period. Bottles with narrow neck and volume of about 280 mL were filled with the sample and positioned on a magnetic stirrer. Then, the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottleneck to avoid contact of the sample with the atmosphere.
The decline of oxygen concentration was measured and recorded at min. 10-minute intervals.
The pH was determined in the remaining part of the reaction mixture.


TEST CONDITIONS
Lighting: daily
Stirring and aeration: with filtered pressured air, airflow rate 0.8 L/min.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorphenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

See table No.3 and No.6

Results with reference substance (positive control):

Results with reference substance valid: yes
The EC50 value of the reference substance 3,5-dichlorophenol was: EC50 = 12.2 mg.L-1 (95 % confidence interval: 10.2 – 15.5 mg.L-1).

Any other information on results incl. tables

Main test

The order of test mixtures:

-       control experiment, 1 (K1)

-       5 concentrations of reference substance, 5.2, 7.2, 10, 14 and 19 mg.L^-1, (R1 – R5)

-       5 concentrations of test substance, 100, 180, 320, 560 and 1000 mg.L^-1,(Z1 – Z5)

-       control experiment 2, (K2)

-       abiotic decomposition at the highest concentration of test substance, 1000 mg.L^-1, (Zab)

Table 3: Performance of the main test

Container No.	Label	Test/Ref. substance  dose /500 mL	Test/Ref. substance concentration mg.L^-1	Medium dose   mL	Sludge dose   mL	Water dose   mL	pH     start	pH     end
1	K 1	0	0	16	200	up to 500 mL	7.08	7.63
2	R 1	19 mL	19	16	200	up to 500 mL	7.12	7.85
3	R 2	14 mL	14	16	200	up to 500 mL	7.10	7.84
4	R 3	10 mL	10	16	200	up to 500 mL	7.09	7.76
5	R 4	7.2 mL	7.2	16	200	up to 500 mL	7.08	7.69
6	R 5	5.2 mL	5.2	16	200	up to 500 mL	7.06	7.69
7	Z 1	50 mg	100	16	200	up to 500 mL	7.04	7.62
8	Z 2	90 mg	180	16	200	up to 500 mL	7.03	7.65
9	Z 3	160 mg	320	16	200	up to 500 mL	7.01	7.62
10	Z 4	280 mg	560	16	200	up to 500 mL	6.99	7.61
11	Z 5	500 mg	1000	16	200	up to 500 mL	6.94	7.58
12	K 2	0	0	16	200	up to 500 mL	7.09	7.68
13	Bab	500 mg	1000	16	0	up to 500 mL	7.13	7.04

Table 6: Evaluation of inhibition of respiration in the main test

Containernumber	Label	Conc. mg.L^-1	p1 mg.L^-1	p2 mg.L^-1	t1 min	t2 min	R mg.L^-1. h^-1	Inhibition %
1	K 1	0	6.50	2.65	0.5	7.5	33.00	0
2	R 1	19	8.02	6.48	0	10	9.24	71.4
3	R 2	14	7.88	5.71	0	10	13.02	59.8
4	R 3	10	7.44	4.08	0	10	20.16	37.7
5	R 4	7.2	7.28	2.95	0	10	25.98	19.7
6	R 5	5.2	6.45	2.51	1.0	9.5	27.81	14.1
7	Z 1	100	6.37	2.47	0.5	7.5	33.43	-3.3
8	Z 2	180	6.39	2.52	1.5	8.5	33.17	-2.5
9	Z 3	320	6.44	2.60	0.5	7.5	32.91	-1.7
10	Z 4	560	6.43	2.63	1.0	8.0	32.57	-0.6
11	Z 5	1000	6.50	2.69	0	7.0	32.66	-0.9
12	K 2	0	6.37	2.67	1.5	8.5	31.71	0
13	Zab	1000	8.66	8.69	0	10	-0.18	-

Average value of K1, K2 = 32.36 mg.L^-1. h^-1,     SD = 0.912 mg.L^-1. h^-1

EC values for test substance, Ashes (residues)

In the main test Ashes (residues) caused no inhibition of the sludge respiration (see table 6). Therefore it was not possible to calculate the EC₅₀(EC₂₀, EC₈₀) value from the obtained respiration rates.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Ashes (residues), the EC50 was found to be: EC50 > 1000 mg.L-1
Under the current experimental conditions, all tested concentrations in the main test provided no inhibition of respiration rate (see table 6). From available data it was not possible to calculate EC50 value.

Executive summary:

The influence of the test substance, Ashes (residues), on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

Test performance

Test was performed according to:

Method C.11 - Activated Sludge Respiration Inhibition Test, Directive 88/302/EEC. Published in OJ L 133 1988.

The preliminary test was performed using 5 concentrations in range 46 - 1000 mg.L-1 with a geometric factor of 2.2 to determine the concentrations to be used in the main test.

In the main test, 5 concentrations of the test substance in geometric progression with factor of 1.8 (100, 180, 320, 560, 1000 mg.L-1) were used together with two control experiments (without the test substance), one at the beginning and one at the end of the test.

3,5-Dichlorophenol was used as reference substance. Five concentrations in geometric progression with a factor of 1.4 (5.2, 7.2, 10, 14 and 19 mg.L-1) were selected.

The abiotic decomposition was not detected even at the highest concentration of the test substance.

The pH value during the main test was in the range of 6.9 to 7.9.

Validity of test

The test met the following validity criteria:

- the respiration rates of two control experiments should not differ from each other by more than 15 %

- the EC50 value of the reference substance should be in the range of 5 – 30 mg.L-1

The difference of two respiration rates of control experiments at the beginning and the end of the main test from each other was:

1.29 mg.L-1.h-1 = 4.07 % (if the value of lower control 31.71 mg.L-1.h-1 = 100 %)

The 3h-EC50 value of reference substance, 3,5-dichlorophenol: EC50 = 12.2 mg.L-1

(95 % confidence interval: 10.2 – 15.5 mg.L-1)

Since all criteria for acceptability of the test were met, this study is considered to be valid.

Results of the respiration inhibition test

On the basis of the effective concentrations describing the respiration inhibition of activated sludge caused by the test substance, Ashes (residues), the EC50 was found to be: EC50 > 1000 mg.L-1

Under the current experimental conditions, all tested concentrations in the main test provided no inhibition of respiration rate (see table 6). Therefore it was not possible to calculate the EC50 value.