Reaction products of 1,4-cyclohexanedimethanol with propylene oxide and ammonia

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:

2014-01-13 - 2014-01-13

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In the range finding test activated sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dissolved directly in water. An amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approx. 5 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 ml) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volum of 500 mL to give the required concentration of 10 mg/L. Similarly, aliquots (200 ml) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several time to ensure homogeneity of the stock solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant a Loughbourough Leicestershire, UK which treats predominantly domestic sewage. A synthetic sewage was added to each test vessel to act as an respiratory substrate.
- Preparation of inoculum for exposure: the activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximatly 21°C and was used on the day of collection. The pH of the sample was 7.9 using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of suspended solids level of the activated sewage sludge was carried out by filtering a sample (100mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approx 105°C for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

approx. 21°C

pH:

Control: 7.3-7.5
10 mg/L: 7.4-7.5
100 mg/L: 7.5
1000 mg/L: 7.5-8.0

Dissolved oxygen:

Control (initial O2): 5.1-5.4 mg O2/L
10 mg/L (initial): 4.7 mg O2/L
100 mg/L: 5.3 mg O2/L
1000 mg/L: 6.7-7.5 mg O2/L.

Nominal and measured concentrations:

nominal : control- 10 -100 -1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: fill volume of 500 mL, no other details reported
- Aeration: yes
- No. of vessels per concentration (replicates): 3 for 1000 mg/L and 1 for 10 and 100 mg/L
- No. of vessels per control (replicates): 2
- No. of vessels per concentration of reference substance (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis water
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and temperature (with mercury-in-glass thermometer) were measured at start and end, respiration rate was measured at end of exposure

OTHER TEST CONDITIONS
- Adjustment of pH: yes, prior to start of exposure (to 7.0)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate, measured after 3 hours in glass samples tubes into which microcathode oxygen electrodes were inserted, and expressed as mg/L/h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding
- Test concentrations: 10, 10, 1000
- Results used to determine the conditions for the definitive study: no (it was decided not to perform a definitve study)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

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:

EC50

Effect conc.:

>= 100 - <= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- EC20(3h) > 100 mg/L
- EC80(3h) > 1000 mg/L
- Less then 10 % inhibition was observed in all test flasks.

Results with reference substance (positive control):

The validation criterion for the reference item EC50 values was also satisfied: EC50 was 8.2 mg/L 3.5-dichlorophenol (CI: 6.4-11). The EC50 of the 3.5- dichlorophenol lies within range 2-25 mg/L.

Reported statistics and error estimates:

The % inhibition values were plotted against concentration for the reference item only, a line fitted using the XIfit software packages and the ECx values determined from the equation from the fitted line. The ECx values for the test item were determined by inspection of the inhibition of respiration rate data. 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon. One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's mutiple comparision procedure for comparing several treatments with a control was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analysis were performed using SAS computer software package.

Table 1: Observations on the test preparations throughout the test period in the range-finding test

Nominal concentrations	Observations on test preparations
	0 hour	30 minutes contact time	3 hours contact time
control (all replicates)	pale yellow/brown dispersion	dark brown dispersion	dark brown dispersion
10 mg/L	pale yellow/brown dispersion, no undissolved test item visible	dark brown dispersion,  no undissolved test item visible	dark brown dispersion,  no undissolved test item visible
100 mg/L	pale yellow/brown dispersion, no undissolved test item visible	dark brown dispersion,  no undissolved test item visible	dark brown dispersion,  no undissolved test item visible
1000 mg/L (all replicates)	pale yellow/brown dispersion, no undissolved test item visible	dark brown dispersion,  no undissolved test item visible	dark brown dispersion,  no undissolved test item visible

Table 2: Oxygen consumption rates and percentage inhibition values after 3 hours contact time in the range-finding test.

nominal concentration	initial O2 reading (mg O2/L)	measurement period (minutes)	final O2 reading (mg O2/L)	O2 consumption rates (mg O2/L/hour)	% inhibition
control R1	5.1	4	2.2	43.5	-
control R2	5.3	5	2.0	39.60	-
control R3	5.3	5	2.1	38.40	-
control R4	5.4	5	2.2	38.40	-
10 mg/L	4.7	4	2.0	40.50	1
100 mg/L	5.3	5	2.1	38.40	4
1000 mg/L R1	6.7	10	3.3	20.40	49
1000 mg/L R2	7.5	10	4.7	16.80	58
1000 mg/L R3	6.7	10	3.5	19.20	52

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-hour EC50 value of between 100 and 1000 mg/L.

Executive summary:

No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects (p <0.05) were shown at the test concentration of 1000 mg/L. Based on these results it was considered unnecessary to perform a definitive test in order to obtain a definitive NOEC and/or an EC50 value for the test item.

Description of key information

In this study, the acute toxicity of the test substance to domestic sewage sludge was investigated in a 3-h respiration inhibition study. The EC50 ranged between 100-1000 mg/L.  The EC10 was > 100 mg/L. A PNECstp was derived from the (unbound) EC10 value.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

A 3 -h respiration inhibition study (Best, 2014) was conducted according to OECD guideline 209. The study was considered as key study and used for PNEC derivation. The nominal preliminary test concentrations were 0, 10, 100 and 1000 mg/L. The dissolved oxygen (DO) concentrations after 30 minutes contact time in the 100 mg/L and 1000 mg/L vessels were below the required 60 -70% of saturation as recommended in the Test Guideline however given that there was no significant inhibition respiration in the 100 mg/L test vessel after 3 hours, the low DO levels were not considered to have contributed to the observed toxicity. As no statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, it was considered unnecessary to perform a definitive study. The effect of the test item gave a 3 -hour EC50 value between 100 and 1000 mg/L.