Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), 2-propenoate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 6, 2010 to April 27, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to the section 13 for details on the read across justification. The toxicity to microorganisms study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

In contrast to the guideline, it was decided to use copper (II) suphate pentahydrate instead of the prescribed 3,5-dichlorophenol. The usage of dichlorophenol is unsuitable for routine lab use.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Preparation and application of test solutions (especially for difficult test substances)
- Method: Direct weighing and stirring for 24 hours
- Eluate: Synthetic waste water according to OECD 209
- Differential loading: 100, 180, 320, 580 and 1000 mg/L
- Controls: 2 control samples without test substance were included, one at the beginning and one at the end of the test.

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: non adapted activated sludge from the sewage plant at Sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: washed twice with autoclaved tap water and adjusted to a dry weight concentration of ca. 4.0 g/L
- Final dry sludge concentration: 4.12 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.8 °C

pH:

6.72

Dissolved oxygen:

-

Salinity:

Not determined

Conductivity:

-

Nominal and measured concentrations:

0, 100, 180, 320, 580 and 1000 mg/L (nominal)

Details on test conditions:

Test system
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Composition of test medium: 200 mL inoculum + 16 mL synthetic waste water + 284 mL autoclaved tap water + test substance concentration (directly weighed out in each test vessel)

Effect parameters measured (with observation intervals if applicable): determination of respiration rates after an incubation period of 3h

Test concentrations
- Based on a range finding study and inhibitions in the preliminary test
- Nominal test substance concentrations:
Concentration [mg/L] Inhibition [%]
10 -1
100 6
1000 43

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

292 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.:

> 1 000 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:

other: EC80

Effect conc.:

> 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:

Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

Results with reference substance (positive control):

Results with reference substance valid? Yes
Relevant effect levels. EC50: 115 mg/L

Reported statistics and error estimates:

The EC20-value of the test substance was calculated by sigmoidal dose-response regression using the software GraphPad Prism4, GRAPHPAD SOFTWARE, INC. The EC50 and EC80-values could not be determined since these values were outside the tested concentration range. The EC50-value of the reference substance was calculated by probit analysis using software SIGMAPLOT (Windows), SPSS CORPORATION. Probit values were taken from WEBER (1986).

Calculations:

From the oxygen depletion, the respiration rates were calculated in mgO₂/(L×h). The inhibition of the respiration rates from the test and reference substance concentrations in % was calculated in comparison with the respiration rates from the control.

Table 1. Respiration rates and inhibition of respiration of test and reference substance concentrations

	Concentration	Respiration rate	Inhibition
	[mg/L]	[mgO2/(L×h)]	[%]
Test item		1000		17.6		46
		580		21.2		35
		320		25.6		21
		180		27.6		15
		100		30.4		6
Control 1		–		32.0		–
Control 2		–		32.8		–
Reference item		180		10.8		67
		100		18.4		43
		58		23.6		27

Table 2. EC-values with confidence interval of the test substance

	EC-values	Confidence interval
	[mg/L]	P = 95 [%] [mg/L]
EC20	292	-
EC50	> 1000	n.d.
EC80	> 1000	-

Table 3. Raw data and respiration rates of test substance, control and reference substance

	Concentration	Oxygen-concentration*	Measured distance**	Respiration rate
	[mg/L]	[mgO2/L]	[mm/3 min]	[mgO2/(L×h)]
Test item	1000	9.04	44	17.6
	580	7.92	53	21.2
	320	6.89	64	25.6
	180	6.67	69	27.6
	100	6.53	76	30.4
Control 1	–	5.62	80	32.0
Control 2	–	6.95	82	32.8
Reference item	180	8.66	27	10.8
	100	7.16	46	18.4
	58	6.60	59	23.6

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 3 h EC20, EC50 and EC80 for the test substance were determined to be 292, >1000 and >1000 mg/L, respectively (in activated sludge of a municipal sewage treatment plant).

Executive summary:

A study was conducted to determine the toxicity to microorganisms of the read across substance, TMP(EO)TA, according to OECD Guideline 209 (respiration inhibition test), in compliance with GLP. Activated sludge, non-adapted (dry sludge concentration of 4.12 g/L) were exposed for 3 h to the test substance at concentrations of 0 (negative control), 100, 180, 320, 580 and 1000 mg/L. Copper (II) sulphate pentahydrate p.a. was used as positive control and gave an EC50 of 115 mg/L. The test was considered valid. The EC20-value of the test substance was calculated by sigmoidal dose-response regression using the software GraphPad Prism4. The EC50 and EC80-values could not be exactly determined since these values were outside the tested concentration range. Based on the results of the read across study, the 3 h EC20, EC50 and EC80 for the test substance were determined to be 292, >1000 and >1000 mg/L, respectively (Fiebig, 2010).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

A study was conducted to determine the toxicity to microorganisms of the read across substance, TMP(EO)TA, according to OECD Guideline 209 (respiration inhibition test), in compliance with GLP. Activated sludge, non-adapted (dry sludge concentration of 4.12 g/L) were exposed for 3 h to the test substance at concentrations of 0 (negative control), 100, 180, 320, 580 and 1000 mg/L. Copper (II) sulphate pentahydrate p.a. was used as positive control and gave an EC50 of 115 mg/L. The test was considered valid. The EC20-value of the test substance was calculated by sigmoidal dose-response regression using the software GraphPad Prism4. The EC50 and EC80-values could not be exactly determined since these values were outside the tested concentration range. Based on the results of the read across study, the 3 h EC20, EC50 and EC80 for the test substance were determined to be 292, >1000 and >1000 mg/L, respectively (Fiebig, 2010).