Fatty acids, C18-unsatd., phosphates

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:

26th June-17th September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

results of toxicity control

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.

Analytical monitoring:

yes

Vehicle:

not specified

Details on test solutions:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
- Solubilising agent (type and concentration if used): No
- Test temperature: 22.0 – 23.1 °C
- pH: 7-6-7.8. At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
- Suspended solids concentration: The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge.
- Other: During the test period, the test media were aerated and stirred continuously.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Pre-treatment: The freshly obtained sludge was used immediately. The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (44 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Test type:

not specified

Water media type:

other: Milli-RO water

Total exposure duration:

14 d

Test temperature:

22.0 – 23.1 °C

pH:

pH: 7-6-7.8

Nominal and measured concentrations:

The test item was tested in duplicate at a target concentration of 21 mg/L

Details on test conditions:

TEST CONDITIONS
- Composition of medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
- Solubilising agent (type and concentration if used): No
- Test temperature: 22.0 – 23.1 °C
- pH: 7-6-7.8. At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.
- Suspended solids concentration: The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge.
- Other: During the test period, the test media were aerated and stirred continuously.

TEST SYSTEM
-Test duration: 28 days for the inoculum blank and test item (last CO2 measurement on day 29). 14 days for the positive and toxicity control (last CO2 measurement on day 15). During the test period, the test media were aerated and stirred continuously.
-Test vessels: 2 litre brown coloured glass bottles.
-Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
-Stock solutions of mineral components
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl Dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
-Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
-Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
-Synthetic air (CO2 < 1 ppm) A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
-Illumination: The test media were excluded from light.

PROCEDURE AND SAMPLING
-Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

-Type and number of bottles.
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, reference item and inoculum (1 bottle).
-Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components. The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
-Experimental CO2 production. The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
-Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test item. Titrations for the positive an

Reference substance (positive control):

yes

Remarks:

A solution of CH3COONa was prepared by dissolving 799.77mg in Milli-RO & making this up to a vol. of 200mL. Vols. of 20mL from it were added to 2l of the test med.of the + control bottle and the toxicity control bottle, final conc.of 40mg CH3COONa per l.

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

In the toxicity control, more than 25% biodegradation occurred within 14 days (54%, based on ThCO2). See details on section "Any other information on results incl. tables." Biodegradation of the positive control (sodium acetate) and toxicity control are presented in figure 1.

Results with reference substance (positive control):

1. The positive control item was biodegraded by at least 60% (85%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 5%).
3.The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (50.6 mg CO2 per 2 litres of medium, corresponding to 25.3 mg CO2/L).
4.The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
Since all criteria for acceptability of the test were met, this study was considered to be valid.

Table 1. CO₂Production and Percentage Biodegradation of the Toxicity Control

Day	HCl (0.05 N) titrated (mL)		Produced CO2 (mL HCl)	Produced CO2 (mg)	Cumulative CO2 (mg)	Biodegradation^1) (%)
	Blank (mean)	Toxicity control
1	47.67	48.37	0.00	0.0	0.0	0
4	46.46	25.53	20.93	23.0	23.0	13
6	47.35	30.97	16.38	18.0	41.0	23
8	47.04	31.97	15.07	16.6	57.6	33
11	47.33	33.67	13.66	15.0	72.6	42
15^2)	46.48	26.38	20.10	22.1	94.7	54
^1): Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test item and positive control: 174.7 mg CO2/2L (ThCO2test item: 89.1 mg CO2/2L + ThCO2sodium acetate: 85.6 mg CO2/2L). ^2): CO2measured on day 15 is actually part of CO2production of day 14, since microbial activity was ended on day 14 by addition of HCl.

Validity criteria fulfilled:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Conclusions:

The toxicity of Fatty acids, C-18-unsatd., phosphates to microorganisms was investigated during a ready biodegradation study. The test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 21 mg/l.

Executive summary:

The toxicity Fatty acids, C-18-unsatd., phosphates to microorganisms was investigated during a ready biodegradation study (CO2 Evolution Test) according to OECD guideline 301 B over a period of 14 days and using an inoculum obtained from activated sludge from a predominantly domestic sewage treatment plant. Inoculum blank, procedural/functional control with the reference substance Sodium acetate and a toxicity control with 21 mg/L test item and 21 mg/L reference item Sodium acetate were performed.

In the toxicity control, more than 25% biodegradation occurred within 14 days (54%, based on ThCO2).

Therefore, the test was found not to inhibit microbial activity in the toxicity control and the tested concentration of 21 mg/L was used as NOEC.

Description of key information

The biodegradation study was conducted for the test item according to OECD 301 B "Ready Biodegradability: CO2 Evolution Test". The test fulfilled all validity criteria mentioned in OECD 301 B guideline and it was conducted in compliance with GLP. Following the "Chapter R.7b: Endpoint specific guidance, Version 4.0 -June 2017" the information content of ready biodegradability (toxicity control) was used to derive a NOEC. The tested concentration was found not to inhibit microbial activity in the toxicity control and the value 21 mg/L was used as NOEC.

The key value for CSA was selected based on the result from the OECD 301B, the tested concentration was used as NOEC.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

21 mg/L