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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
June 14th, 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.
The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted on 4 April 1984
Principles of method if other than guideline:
The study was conducted in accordance with ISO/IEC 17025
GLP compliance:
no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Inoculum: activated sludge from the aeration tank of a municipal biological waste water treatment plant, not adapted, not preconditioned.
- Source: ARA Werdhölzli, CH-8048 Zürich
- Preparation of inoculum for exposure: if sludge was used on the following day, 50 ml of the synthetic sewage concentrate were added per litre of activated sludge and the sludge aerated overnight. Prior to the test, the sludge was washed twice with tap water.
- Pretreatment: activated sludge was used immediately after sampling from the treatment plant without adaptation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
22 ± 0.5 °C
pH:
7.5 ± 0.5
Nominal and measured concentrations:
Nominal: 125, 250, 500, 1000 and 2000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 ml flasks.
- Material, size, headspace, fill volume: a total volume of test suspension of 500 ml.
- Aeration: areation by shaking at 120 rpm for the exposure period.
- Organisms amount per vessel: 1.6 mg/l dry matter in the final mixture.
- No. of vessels per concentration: 1 replicate sample containing activated sludge, test medium and test substance.
- No. of vessels per blank control: 2 replicates with inoculated sample without test substance.
- No. of vessels per reference compound: 1 replicate sample containing activated sludge, test medium and reference compound.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic sewage feed was prepared with tap water. 16 ml of 100-fold sewage concentrate per 500 ml of the final test mixture.
- Composition of the synthetic sewage feed:
peptone 16 g
meat extract 11 g
urea 3 ng
sodium chloride 0.7 g
calcium chloride dihydrate 0.4 g
magnesium sulphate heptahydrate 0.2 g
dipotassium hydrogen phosphate 2.8 g
- pH: 7.1 - 7.4
- Conductivity (20 °C): range 510 - 585 µS/cm
- Total hardness: range 21.1 - 30.2 °F; CO3 hardness range 18.6 - 27.2 °F; rest-hardness range 2.5 - 3.8 °F
- Total hardness: 2.11 - 3.02 mmol/l
- Acid consumption: range 3.72 - 5.44 mmol/l
- KMnO4 consumption: range 0.6 - 1 mg/l
- E. Coli: 0 per 100 ml
- Colony counts: 0 ml at 20 °C; 8 ml at 30 °C
- Ammonium: < 0.01 mg/l
- Nitrite: < 0.002 mg/l
- Nitrate: range 7.5 - 10.5 mg/l
- Chloride: range 4.5 – 14 mg/l
- Phosphate as PO4-P: < 0.002 - 0.023 mg/l
- Sulfate: range 18.5 - 31 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: the pH-value was checked and adjusted to pH 7.5 ± 0.5 with NaOH or H2SO4, if necessary.

EFFECT PARAMETERS MEASURED
The oxygen concentration, in mg/l, was measured with a oxygen electrode (ORION 9708) instead to a 300 ml BOD bottle and recorded for about 5 to 10 min.
The first sample (blank control) was analyzed 3 hours after starting the test followed by the analysis of the test suspension at 10 - 15 min intervals.
Finally, the second blank control was analyzed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 2 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
No significant toxic effects to the activted sludge was recorded. The respiration rates were in the same range as determined for the two blank controls.

The variation between the two controls was ± 2.6 %, therefore the test was considered valid.
Results with reference substance (positive control):
The EC50 was estimated to be 3.7 mg/l, which was in the range of 5 mg/l, thus confirming suitability of activated sludge and test conditions.

Activated sludge respiration rates after 3 h of exposure to the test material and calculation of the inhibition

 

Nominal concentration
(mg/l)
Substance O2consumption rates
(mg O2/l/h)
Respiration inhibition (%)
0 Blank control 43.5
0 Blank control 41.3
125 Test substance 40.3 4.9
250 Test substance 41.3 2.5
500 Test substance 39.4 7.0
1000 Test substance 40.4 4.6
2000 Test substance 39.8 6.2
3 Reference compound 23.8 43.9
10 Reference compound 11.4 73.2
30 Reference compound 4.5 89.5
Validity criteria fulfilled:
yes
Remarks:
the blank controls oxygen uptake was more than 20 mg/l/h and the coefficient of variation in control replicates was ± 2.6 %; the EC50 of 3,5-DCP was in the range 2 - 25 mg/l using activated sludge from domestic sewage
Conclusions:
EC50 (3h) > 2000 mg/l (nominal)
Executive summary:

The inhibitory effect of the test substance on oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions, according to the OECD Guideline 209. Nominal concentrations of the test substance were 125, 250, 500, 1000 and 2000 mg/l, respectively. The calculation of the EC20, EC50 and EC80 was performed using the nominal concentration values, assuming the test substance to be stable in water over 3 hours.

At the tested concentrations, no significant toxic effects were shown to the activated sludge. Respiration rates were in the same range as determined for the two blank controls. The EC50 of the reference compound was estimated to be in the specific range, confirming suitability of activated sludge and test conditions. The variation between the two controls was ± 2.6 %, therefore the test was considered valid.

Conclusion

EC50 (3h) > 2000 mg/l (nominal)

Description of key information

EC50 (3 h) > 2000 mg/l (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:
2 000 mg/L
EC10 or NOEC for microorganisms:
2 000 mg/L

Additional information

The existing data on Direct Blue 199 sodium salt (i.e. Direct Blue 199_Na) have been taken into account in order to assess the potential for toxicity to microorganisms of Direct Blue 199. The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point. The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.

 

The inhibitory effect of Direct Blue 199_Na on oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions, according to the OECD Guideline 209. Nominal concentrations of the test substance were 125, 250, 500, 1000 and 2000 mg/l, respectively. At the tested concentrations, no significant toxic effects were shown to the activated sludge. Respiration rates were in the same range as determined for the two blank controls. The EC50 of the reference compound was estimated to be in the specific range, confirming suitability of activated sludge and test conditions. The variation between the two controls was ± 2.6 %, therefore the test was considered valid.

 

The outcomes of the key study are confirmed by further other studies conducted both in bacteria and sludge. Despite many detail are missing, the results can be considered in a supporting approach. The inhibition concentration resulted higher than 100 and 40 mg/l; the lowest concentration which clearly inhibits bacterial growth or sewage sludge (BST and KST), based on visible opacity of the test solution, resulted in all cases higher than 300 mg/l.

 

In conclusion, Direct Blue 199 can be considered as non harmful/toxic to microorganisms.