Dapsone

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:

26 August 2011-26 August 2011

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The batch of 4,4’-DDS tested was a white to off white powder with a purity of 98.3 Area (%). The test substance was not completely soluble in test medium at the loading rate initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24-26 hours. Subsequently, 16 ml synthetic sewage feed, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

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 (3.0 g/l of sludge, as used for the test). The pH was 7.9 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

Remarks on exposure duration:

during which aeration and stirring took place

Post exposure observation period:

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

Test temperature:

Between 20.5 and 22.0°C.

pH:

pH before addition of sludge: 7.6-7.7.
pH after the 3 hour exposure period: 7.5-8.5.

Dissolved oxygen:

The oxygen concentration at the start of exposure was at least 7.2 mg O2/l.

(Mean) Respiration Rates:
Control: 35 mg O2/l.h
T1 (a loading rate of 10 mg/l): 31 mg O2/l.h
T2 (a loading rate of 100 mg/l): 32 mg O2/l.h
T3 (a loading rate of 1000 mg/l): 25 mg O2/l.h

Nominal and measured concentrations:

Nominal concentrations, three loading rates: 10, 100 and 1000 mg/l.

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass
- Aeration: During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 10 mg/l (1), 100 mg/l (1), 1000 mg/l (3).
- No. of vessels per control (replicates): 6
- 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 ionexchange cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).

EFFECT PARAMETERS MEASURED
(with observation intervals if applicable) : 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 and 7 mg O2/l.

TEST CONCENTRATIONS
- Test concentrations: 10, 100 and 1000 mg/l

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The combined limit/range-finding test showed 12 and 9% inhibition of the respiration rate at a loading rate of 10 and 100 mg/l, respectively. A statistically significant inhibition on respiration rate, average value of 29%, was found at a loading rate of 1000 mg/l compared to the control (2 Sample t-Test). Thus, the EC50 was above the highest loading rate tested, a loading rate of 1000 mg/l.

Furthermore, there was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (9.9 mg/l).

Reported statistics and error estimates:

ECx
The percentage inhibition was plotted against the logarithm of the loading rates. The EC50 for the reference substance was determined using linear regression analysis. If available, the 95% confidence limits of the EC values were given. For the test substance the EC50 was above the highest loading rate tested.

NOEC estimation
As discussed with the sponsor, no final test was performed to determine the NOEC.

Combined limit/range-finding test - Study results

Flask	Conc./loading rate (mg/l)	pH before addition of sludge	pH after 3h contact time	Oxygen conc. at the start (~mg 02/l)	Respiration rate (mg 02/l h)	Respiration rate (mg 02/g h)^1	% inhibition of the respiration rate relative to control (mean value)
C1	0	7.7	8.1	7.2	35	24
C2	0	7.7	8.4	8.6	33	22
C3	0	7.6	8.2	7.8	30	20
C4	0	7.6	8.2	8.4	36	24
C5	0	7.6	8.0	8.5	41	28
Mean					35	24
SD					4	3
Coefficient of variation (%)					11	11
C-N1 (with ATU)	0	7.7	8.2	8.0	23	15
R1	5.0	7.7	8.4	8.4	25	16	31
R2	12	7.7	8.3	8.5	16	10	56
R3	30	7.6	8.2	8.7	7	5	80
T1	10	7.6	8.2	8.1	31	21	12
T2	100	7.6	8.1	8.3	32	21	9
T3A	1000	7.6	8.3	8.5	23	16	34
T3B	1000	7.6	8.2	8.7	25	17	28
T3C	1000	7.6	8.2	9.0	27	18	24
T3 Mean					25	17	29
T3 (abiotic)	1000	7.6	7.5	8.9	0	0	99
t3 (with ATU)	1000	7.6	8.5	8.6	17	11	27^2

¹- the amount of suspended solids in the final test mixture was 1.5 g/l

² - relative to C-N1

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test 4,4’-DDS was slightly toxic to waste water (activated sludge) bacteria. The EC50 however was above the highest loading rate tested, a loading rate of 1000 mg/l.

Executive summary:

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007). The batch of 4,4’-DDS tested was a white to off white powder with a purity of 98.3 Area (%). The test substance was not completely soluble in test medium at the loading rate initially prepared. Therefore, the test substance – Milli-RO water mixtures were magnetically stirred for a period of 24-26h. Subsequently, synthetic sewage feed, sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was measured and recorded for approximately 10 minutes.
In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, blank controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest loading rate with a nitrification inhibitor were tested. The combined limit/range-finding test showed 12 and 9% inhibition of the respiration rate at a loading rate of 10 and 100 mg/l, respectively. A statistically significant inhibition on respiration rate, average value of 29%, was found at a loading rate of 1000 mg/l compared to the control. The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid. In conclusion, 4,4’-DDS was slightly toxic to waste water (activated sludge) bacteria. The EC50 however was above the highest loading rate tested, a loading rate of 1000 mg/l.

Description of key information

Under the testing conditions of the activated sludge respiration inhibition test (carbon and ammonium oxidation) with 4,4’-DDS, 4,4’-DDS was slightly toxic to wastewater (activated sludge) bacteria. The EC50 however was above the highest loading rate tested, a loading rate of 1000 mg/l. 

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L