[No public or meaningful name is available]

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:

April 2016 - October 2016

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 (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL:
- Chemical name: Phosphonium, tetrakis (hydroxymethyl)-,sulphate(2:1); polymer with urea
- CAS number: 63502-25-0
- Batch no.: PLFS17D1
- Expiration date of the lot/batch: 31. August 2016
- Purity: Active content (Iodine titration): 67.8% w/w / THPS (CAS number 55566-30-8): 25.7% w/w
- Solubility in water: Miscible
- pH: 5.0 at 20 °C
- Specific gravity (s.g.): 1.377 g/ml
- Appearance: Clear, yellow liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Stability under correct storage conditions (expiry date): 31. August 2016
- Storage requirements: Keep container tightly closed in a cool, dry place.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test concentrations were prepared without using a stock solution. The replicates of the test concentrations were prepared by direct addition of the test item to the mixture of deionised water and synthetic sewage feed just before adding the microbial inoculum.
Before starting the exposure (i.e., adding the microbial inoculum) the pH in the test solutions of the definitive test was 7.1–7.3. An adjustment was therefore not necessary.
A stock solution of 500 mg/l (nominal) was prepared of the reference substance 3,5- dichlorophenol by dissolving 0.5 g 3,5-dichlorophenol in 10 ml of 1N NaOH, diluting to 30 ml with
deionised water, and adding 8.2 ml of 1N H2SO4 under stirring to the point of incipient precipitation. The mixture was diluted to one litre with deionised water. The pH in the stock solution was 7.2.
The various concentrations of the reference item were prepared by diluting the stock solution with deionised water, and subsequently with synthetic feed and inoculum. Respective controls were prepared without the test item.
The total mixture volume for each replicate of the test item concentrations and the controls was 500 ml per vessel. Immediately after addition of the microbial inoculum, the test solutions were aerated. The test vessels were covered with watch-glasses.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The activated sludge was obtained from the sewage treatment works in Flörsheim/Main (Germany) treating predominantly domestic sewage.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

Range from 19.8 to 20.4°C (n= 80)

pH:

Range from 7.1 to 7.3

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Control; 250, 79.1, 25.0, 7.91 and 2.50 mg test item/L (nominal) , these concentrations correspond to 169.5, 53.6, 17.0, 5.36 and 1.70 mg active ingredient/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 ml glass beaker with watch-glass cover
- Aeration: air flow of 50–60 l/h
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Maintenance time of the activated sludge before use: Two days
- pH-value of the activated sludge: 7.5
- Test vessels: 1000 ml glass beakers (sterilised in a drying oven at 150 °C for 3 hours)
- Volume of test mixture per test vessel: 500 ml
- Volume of microbial inoculum per test mixture: 200 ml
- Volume of synthetic sewage feed per test mixture: 16 ml
- Mixed liquor suspended solids level in the microbial inoculum on testing day: 4.069 g/l
- Mixed liquor suspended solids level in the final test mixtures: 1.628 g/l (within 1.5 g/l ±
10%), calculated as: Mixed liquor suspended solids level in the microbial inoculum [g/l]×Volume ofmicrobial inoculum per test mixture [ml]/volume of test mixture per test vessel [ml]
- Nitrification inhibitor used : N-allylthiourea in the preliminary rangefinding test


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Particulate matter:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:
- Details on termination of incubation:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using fewer concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol, CAS 591-35-5 , purity: 99.7%

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

71.06 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

104.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

19.85 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

29.28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

1) Range-finding Test:
The non-GLP range-finding test including a nitrification inhibition control showed an inhibition of total respiration of 100% compared to the controls at 1000 mg test item/l, of 36.1% inhibition
at 100 mg test item/l, and no inhibition at 10 mg test item/l. A specific inhibition of nitrification by the test item was not observed in any treatment of the rangefinder. The definitive test was therefore performed without ATU.

2) Definitive test:
The definitive test was therefore performed without investigating the inhibition of nitrification.
The definitive test showed a clear concentration-response relationship with an inhibition of total respiration rate of up to 77% of control respiration.
The main results of the definitive test are summarised in the following tables in "any other information on results including tables"

Results with reference substance (positive control):

Four concentration levels (1.8, 5.9, 18.8, 60 mg/l) were tested.
The EC50 (3 hours) of 3,5-dichlorophenol was 7.9 mg/L.
This reference test were within the ranges accepted by the test guideline (5-25 mg/L) and thereby confirm appropriate test conditions.

Reported statistics and error estimates:

results were performed using the Probit analysis. Computation of variances and confidence limits was adjusted to metric data (Christensen & Nyholm 1984). Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.9, respectively.

2) defintive test:

Table 1: : Decrease of total respiration rate (inhibition in % of control) as caused by the test item after 3 h. :

Treatment [mg test item/l]	Treatment [mg active ingredient/l]	mean respiration rate [mg O2/(l*h)]	standard deviation	number of replicates	inhibition of respiration rate [% of control value]
Control	Control	36.67	5.8155	6	0.0
2.50	1.70	48.12	13.9057	3	-31.2
7.91	5.36	58.63	2.7923	3	-59.9
25.0	17.0	44.42	15.8915	3	-21.1
79.1	53.6	20.40	1.3992	3	44.4
250	169.5	8.43	0.9294	3	77.0

Table 2: Selected effective concentrations (EC_x) of the test item and the active ingredient, and their 95%- confidence limits (according to Fieller's theorem):

Parameter	EC10	EC20	EC50	EC80
	test item
Value [mg /l]	29.28	45.36	104.81	242.17
lower 95%-cl	5.72	14.53	67.80	156.10
upper 95%-cl	50.25	69.61	165.57	798.09
	active ingredient
Value [mg /l]	19.85	30.75	71.06	164.19
lower 95%-cl	3.88	9.85	46.0	105.8
upper 95%-cl	34.1	47.2	112.3	541.1

Validity criteria fulfilled:

yes

Conclusions:

The definitive test showed a clear concentration-response relationship with an inhibition of total respiration rate of up to 77% of control respiration. The EC50 (3h) was determined to be 104.81 mg/L as test item (nominal) that correspond to 71.06 mg/L expressed as Active ingrédient (using the content of active ingredient of 67.8% (w/w)).

Executive summary:

The effect of the test item on the respiration of bacteria in activated sludge was investigated in a static test system. The respiration rate of the test system was measured after an exposure period (contact period) of 3 hours. The study was performed according to the OECD Guideline No. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)" and in compliance with the GLP.

In addition the effect of the test item on the respiration of nitrifying bacteria in activated sludge was investigated in a preliminary non-GLP range-finding test.

This preliminary non-GLP range-finding test including a nitrification inhibition control showed a clear inhibition of total respiration. However, a specific inhibition of nitrification-related respiration was not observed in any treatment of the rangefinder.

The definitive test was therefore performed without ATU and according to the results of this total respiration inhibition the following five concetrations were tested ;250, 79.1, 25.0, 7.91 and 2.50 mg test item/l.

These concentrations correspond to the following concentrations of active ingredient, using the content of active ingredient of 67.8% (w/w) ; 169.5, 53.6, 17.0, 5.36 and 1.70 mg active ingredient/L.

The definitive test showed a clear concentration-response relationship with an inhibition of total respiration rate of up to 77% of control respiration.

The EC50 (3h) was determined to be 104.81 mg/L as test item (nominal) that corresponds to 71.06 mg/L expressed as Active ingredient.

Description of key information

The effect of the test item on the respiration of bacteria in activated sludge was investigated in a static test system and was measured after an exposure period (contact period) of 3 hours. The study was performed according to the OECD Guideline No. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)" and in compliance with the GLP.

In addition the effect of the test item on the respiration of nitrifying bacteria in activated sludge was investigated in a preliminary non-GLP range-finding test.

The definitive test showed a clear concentration-response relationship with an inhibition of total respiration rate of up to 77% of control respiration.

The EC50 (3h) was determined to be 104.81 mg/L as test item (nominal) that corresponds to 71.06 mg/L expressed as Active ingredient.

Key value for chemical safety assessment

EC50 for microorganisms:

71.06 mg/L

EC10 or NOEC for microorganisms:

19.85 mg/L