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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2010
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 Diammonium hydrogenorthophosphate tested consisted of white crystals with a purity of 99.8%. A stock solution of 0.5 g/L was prepared by adding 0.1252 g test substance to 250 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Thorough mixing (vortex) was used to accelerate dissolution and to ensure homogeneity. Volumes of the stock solution corresponding to the test concentration were then added to the test media. A concentration of 100 mg/L was tested in duplicate. 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 (4.0 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
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours exposure period.
Test temperature:
between 19.9 and 20.0°C
pH:
Between 7.7 and 7.9
Dissolved oxygen:
Oxygen consumption:
Controls test substance series:67-71 mg O2/L/h
Test substance series: 66-69 mg O2/L/h
The oxygen concentration at the start of measurements was at least 6.5 mg O2/l
Nominal and measured concentrations:
Nominal concentration: 100 mg/L in duplicate
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass, approximately 300 mL oxygen bottles (measuring) and
1 litre test bottles(exposure)
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates):2
- No. of vessels per control (replicates):2/ series
- Biomass loading rate:1.6 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)
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 2.5 and 6.5 mg O2/L.

TEST CONCENTRATIONS
- Test concentrations: limit study at 100 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 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 inhibition of respiration rate of the sludge was recorded at 100 mg Diammonium hydrogenorthophosphate per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/L (based on nominal concentrations).
Results with reference substance (positive control):
Results with reference substance valid: Yes
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing concentration , ranging from a 43 % inhibition at 5,0 mg/l to 87% at 30 mg/l.
Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 6.0 mg/l
Reported statistics and error estimates:
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For Diammonium hydrogenorthophosphate no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 100 mg/L).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this present test, Diammonium hydrogenorthophosphate was not toxic to waste water (activated sludge) bacteria at 100 mg/L (NOEC). The 3-hour EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/L (based on nominal concentrations).

Description of key information

No reliable study with ammonium dihydrogenorthophosphate is present. In a reliable study with diammonium hydrogenorthophosphate the 3-hr EC-50 was determined to >100 mg/L.  The read-across rationale can be found in the category approach document attached in Section 13 of IUCLID and is fully incorporated in the CSR. 

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

No reliable study with ammonium dihydrogenorthophosphate is present. However, in a reliable OECD 209 and EC C.11 guideline study, the respiration rate of the predominantly domestic sludge was determined after exposure to 100 mg/L diammonium hydrogenorthophosphate for three hours. No significant inhibition of respiration rate of the sludge was recorded at this dose level. The substance was not toxic to waste water (activated sludge) bacteria at 100 mg/L. Therefore, no further testing was needed. Hence, the EC50 of Diammonium hydrogenorthophosphate exceeded 100 mg/L (based on nominal concentrations).