4,10-dibromodibenzo[def,mno]chrysene-6,12-dione

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD- and GLP-Guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: No vehicle, directly weighing, overnight stirring
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 1000 mg/L
- Controls: Test medium without test or reference substance. Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %
- Dry sludge concentration: 3.16 g/L (corresponding to 1.58 g/L suspended solids in the test vessel)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.5-22 °C

pH:

7.87 (activated sludge)
7.84 (synthetic waste water)

Salinity:

None, freshwater

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test or reference item concentration (directly weighed into each test vessel)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control. reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS

- Range finding study
Inhibition of Respiration (Preliminary Tests)
Nominal Test Item
Concentration
[mg/L] Inhibition[%]
1st 2nd
10 3 -
100 -3 -
1000 3 1

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 114 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test was done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
The EC-value of the reference item was calculated by by linear regression using software GraphPad Prism5. GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures.
The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel, MICROSOFT CORPORATION.

EC-Values with Confidence Interval of the test item

	EC-values	Confidence interval
	[mg/L]	P = 95 [%] [mg/L]
NOEC	1000	-
EC20	> 1000	-
EC50	> 1000	n.d.
EC80	> 1000	-

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
                   of Control, Reference and Test Item Concentrations

Reference and Test Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	26.4	16.7	-	-
	2	24.8	15.7
	3	25.2	15.9
	4	25.6	16.2
	5	24.4	15.4
	6	23.6	14.9
58	1	21.6		14	15
	2	20.8		17
	3	21.6		14
100	1	13.6		46	46
	2	12.8		49
	3	14.4		42
180	1	6.4		74	72
	2	7.2		71
	3	7.2		71
1000	1	28.0		-12	-12
	2	27.6		-10
	3	28.4		-14

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is 1000 mg/L. The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the limit concentration 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The inhibition of respiration for the test item replicates ranged from -14 to -10 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 114 mg/L.

Validity criteria of the test guideline were fulfilled.

EC-Values with Confidence Interval of the test item

	EC-values	Confidence interval
	[mg/L]	P = 95 [%] [mg/L]
NOEC	1000	-
EC20	> 1000	-
EC50	> 1000	n.d.
EC80	> 1000	-

The NOEC of the test item is 1000 mg/L.

The test item caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Description of key information

A respiration inhibition test with activated sludge according to OECD 209 (2010) was performed under static conditions with the nominal limit concentration of 1000 mg/L. The test item caused no inhibiting effects at a concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant. The NOEC is 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L