P-1401

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 July 2015 - 30 July 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of inoculum/activated sludge: Aeration tank of Sewage Treatment Plant ”Czajka” , Warsaw. The coarse particles were removed by filtration. Such prepared sludge was washed in the medium and was placed in laboratory-scale unit where the aerobic conditions were maintained by means of an intense aeration with compressor and aerators.
- Preparation of inoculum for exposure: The concentrated sludge was suspended in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated until application. After complete re-suspension was achieved, a sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
- Pretreatment: Inocula was pre-conditioned to the experimental conditions. Pre-conditioning consisted of aerating activated sludge in mineral medium for 2 days at the test temperature of 22 ºC
- Concentration of sludge: 30 mg/litre SS.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

22 ± 2 ºC

pH:

7.4 ± 0.2 ºC

Details on test conditions:

The toxicity to microorganism was assessed within the ready biodegradability test under the following conditions:

TEST CONDITIONS
- Composition of medium:
The following stock solutions were prepared using analytical grade reagents:
(a) Monopotassium dihydrogen orthophosphate, KH2PO4 8.50 g
Dipotassium monohydrogen orthophosphate, K2HPO4 21.75 g
Disodium monohydrogen orthophosphate dihydrate Na2HPO4 · 2 H2O 33.40 g
Ammonium chloride, NH4Cl 0.50 g
Dissolve in water and make up to 1 litre The pH of the solution should be 7.4.
(b) Calcium chloride, anhydrous, CaCl2 27.50 g
Dissolve in water and make up to 1 litre
(c) Magnesium sulphate heptahydrate, MgSO4 · 7 H2O 22.50 g
Dissolve in water and make up to 1 litre.
(d) Iron(III) chloride hexahydrate, FeC13 · 6H2O 0.25 g
Dissolve in water and make up to 1 litre.
Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 11 with dilution water.
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: thermo-cabinet WTW TS 606 CZ-G/3-VAR
- Number of culture flasks/concentration: Flask 10, 11 and 12: Toxicity control: containing test item, reference item at the same concentrations as in the individual solutions and inoculum 30 mg/l SS.
- Measuring equipment: Consumption of oxygen: Closed WTW OxiTop OC 110 respirometer for BOD determination. The consumption of oxygen was determined from the change in pressure in the apparatus. The carbon dioxide, evolved during test item degradation, was absorbed in a solution of potassium hydroxide. Determination of nitrates and nitrites: Spectrophotometer Hach DR 3900 and 45600 reactor COD for nitrate and nitrite concentration measurements. Nitrates were determined using LANGE Cuvette Test method 8039 ( and nitrites were determined using LANGE Cuvette Test LCK341 (ISO 8466-1 and DIN 38402).

SAMPLING
The oxygen uptake from the readings taken at regular and frequent intervals was calculated, using the methods given by the manufacturer of the equipment. The data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recoded and stored in the measuring heads of the sample bottles. These collected data, using the controller, were infrared read out from the heads and stored in the controller.

Reference substance (positive control):

not specified

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Oxygen consumption

Remarks on result:

other: (toxicity assessment within ready biodegradability test)

Details on results:

In the toxicity control containing both the test item and the reference item acetic acid, sodium salt, a mean of 38.5 % biodegradation was noted within 14 days of incubation. The test item can be assumed to be not inhibitory at the applied concentration level of 100 mg/L on the activated sludge microorganisms because degradation was >25 % within 14 days. Based on these results, the NOEC (28d) for the test item was determined to be 100 mg/L.

Validity criteria fulfilled:

not applicable

Conclusions:

The NOEC (28d) for microorganisms of the test item was determined to be 100 mg/L. ity was determined to be 2.5 mg/L (basis for effect: no inhibitory at the highest dose).

Executive summary:

The study on ready biodegradability of test substance in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4-D Method (GLP study). 30 mg/L of activated sludge were exposed to 100 mg/L test item and a reference substance during 28 days to check the possible inhibitory effect. The degradation was followed by the determination of oxygen uptake. As the test substance contains nitrogen, the observed oxygen uptake by the reaction mixture was corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate. On day 14, 38.5% of biodegradation was observed and thereby, test item can be assumed to be not inhibitory to microosganisms at applied concentration of 100 mg/L. Based on these results, the NOEC (28d) for toxicity to microorganisms was determined to be 100 mg/L.

Description of key information

Key study: Test method OECD Guideline 301F. GLP study. The NOEC (28 days) for toxicity to microorganism was determined to be 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Key study: The study on ready biodegradability of test substance in an aerobic aqueous medium with manometric respirometry method was carried out according to OECD 301F and EU C.4-D Method (GLP study). 30 mg/L of activated sludge were exposed to 100 mg/L test item and a reference substance during 28 days to check the possible inhibitory effect. On day 14, 38.5% of biodegradation was observed and thereby, test item can be assumed to be not inhibitory to microosganisms at applied concentration of 100 mg/L. The NOEC (28d) for toxicity to microorganisms was determined to be 100 mg/L.