1,3-di-o-tolylguanidine

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:

2011

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)

Details on test solutions:

Test solution of 3,5 – dichlorophenol.
A 0.5 g/L stock solution of 3,5 – dichlorophenol in ultra-pure- water is used for preparing the different tested solutions from 0.1 to 40 mg/L with factor of 4.5.

N-Allylthiourea solution
Following the guideline, a 2.32 g/L solution of N-allylthiourea (ATU) was prepared. Addition of 2.5 mL of this solution in a final volume of 500 mL gives a final oncentration of 11.6 mg/L (0.1 mM) of ATU known to be sufficient to cause 100 % inhibition of 1.5 g/L of suspended solids.

Introduction of the test item
Usually the different exposure concentrations are prepared by dilution of a stock solution of the test item (range finding test). As the solubility of the test item is very low at the concentrations of the definitive test, in agreement with the sponsor, the WAF method has been performed (i.e. adequate mass of test item was directly added into each test vessel).

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The inoculum comes from the activated sludge of the biological domestic wastewater treatment plant from the city of Abidos (France, 64). The effluent, obtained 2 days before seeding the flasks, is centrifuged at approximately 20 °C for 20 min at 4000 g; the base is re-suspended in ultra-pure water in order to keep the concentration unchanged with respect to the sample. The amount of dry material in the inoculum, is determined by dessiccation at 105 °C until a consta nt weight. From this result the amount of wet sludge can be calculated in order to obtain an activated sludge with a mixed liquor suspended solids level of 4 g/L (+/-10%). The inoculum is preconditioned (aeration). Every day, 50ml/L of synthetic sewage feed are added until the day the flasks are inoculated.
Value of suspended solids was 5.62 g/L. Then a dilution was used to obtain 3 g/L. This level corresponds to a concentration of 1.5 g/L in the test medium.

Total exposure duration:

3 h

Test temperature:

20 +/- 2 °C

pH:

7.7

Nominal and measured concentrations:

Range finding test
A stock solution of DIORTHOTOLYL GUANIDINE was prepared with ultra-pure water by mixing 100 mg of test item in 1 L of water. After 18 h stirring, the mixture was filtered on a cellulose acetate membrane at 0.45 µm. For the range finding test, the final dilutions in the test vessels (in % of dilution) were 46.8, 23.4, 4.7, 0.47 and 0.05.

Definitive test
As barely 15 % inhibition was observed at the higher concentration in the range finding test, the definitive test was performed as WAF from 5 to 100 mg/L of DIORTHOTOLYL GUANIDINE with a factor of 2.115: 5, 10.6, 22.3, 47.3 and 100 mg/L.

Details on test conditions:

The tests are carried out in 500 mL flasks, with the following repartition:
- 16 ml of synthetic sewage feed
- adequate masse of test substance and ultra-pure water for different concentrations
- 250 ml of sludge suspension At T0, the first control (control 1) without item is prepared.

Then every 15 min. the others vessels with different concentrations of item are prepared in turn. Finally, the second control (control 2) is prepared. All suspensions are aerated and stirred during 3 hours and incubated at 20 +/- 2 °C without light. Then a sample of each mixture is poured into a BOD bottle (following the order of preparation). An oxygen electrode is inserted into the bottle. The oxygen concentration is measured every minute during a period of 10 min. The dissolved oxygen concentrations are determined electrochemically using an oxygen electrode and meter.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol CAS number: 591-35-5 EC number 209-714-9 Date of reception in the test facility: 06/07/06 Expiration date of the test item: 07/2011 Batch number/supplier: 15809KI-444 Reference Item characteristics: white odourless granules

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

22.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total Respiration Inhibition (without ATU)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Total Respiration Inhibition (without ATU)

Details on results:

see table below

Results with reference substance (positive control):

see table below

Reported statistics and error estimates:

The respiration rate is calculated from the recorder trace as mg O2/L.h over a time period of 10 min. The portion of the respiration curve over which the respiration rate is measured should be linear. The inhibition effect at each concentration is expressed as percentage of mean of the two control
respiration rates.

Three respiration rates were evaluated : total respiration rate, heterotrophic respiration rate (in presence of N-Allylthiourea) and nitrification respiration rate by difference between total and heterotrophic respiration rates.

The No-Observed-Effect Concentration (NOEC) was determined by statistic analysis using the Dunnett’s test

Respiration rates recapitulation and EC50 for 3,5 -dichlorophenol

	Total Respiration without ATU		Heterotrophic Respiration with ATU		Nitrification Respiration by difference
Control	TR mg/L,h	CV %	HR mg/L,h	CV %	NR mg/L,h	CV %
C1	72.0	2.0	51.6	10.6	20.4	48.4
C2	70.0		60.0		10
Mean	71.0		55.8		15.2
DCP (mg/L)	TR mg/L,h	Inh, %	HR mg/L,h	Inh, %	NR mg/L,h	Inh, %
0.098	72.0	-1.4	56.0	-0.4	16.0	0.00
0.439	72.0	-1.4	55.5	0.5	16.50	-8.55
1.975	72.0	-1.4	48.0	14.0	24.00	-57.89
8.889	61.5	13.4	43.0	22.9	18.50	-21.71
40	10.7	84.9	6.7	88.0	4.00	73.68
EC50 (mg/L)	Total respiration		Heterotrophic respiration		Nitrification respiration
	18.00		12.0		ND

Respiration rates recapitulation and EC50 for DIORTHOTOLYL GUANIDINE

	Total Respiration without ATU		Heterotrophic Respiration with ATU		Nitrification Respiration by difference
Control	TR mg/L,h	CV %	HR mg/L,h	CV %	NR mg/L,h	CV %
C1	82.0	5.4	52.5	19.2	29.5	-
C2	76.0		69.0		-
Mean	79.0		60.8		29.5
DOTG(mg/L)	TR mg/L,h	Inh, %	HR mg/L,h	Inh, %	NR mg/L,h	Inh, %
5	81.0	-2.5	56.0	7.8	25.00	15.30
10.6	87.0	-10.1	58.0	4.5	29.00	1.70
22.3	82.0	-3.8	52.8	13.1	29.20	1.00
47.3	74.0	6.3	45.6	24.9	28.40	3.70
100	52.0	34.2	33.0	45.7	19.00	35.60
EC50 (mg/L)	Total respiration		Heterotrophic respiration		Nitrification respiration
	> 100		> 100		> 100

Conclusions

Total Respiration Inhibition (without ATU1) EC50 – 3h
EC50	Nominal concentrations, mg/L	NOEC, mg/L
3,5-Dichlorophenol	18	-
DIORTHOTOLYL GUANIDINE	> 100	22.0
Heterotrophic Respiration Inhibition (with ATU1) EC50 – 3h
EC50	Nominal concentrations, mg/L	NOEC, mg/L
3,5-Dichlorophenol	12	-
DIORTHOTOLYL GUANIDINE	> 100	5
Nitrification Respiration Inhibition EC50 – 3h
EC50	Nominal concentrations, mg/L	NOEC, mg/L
3,5-Dichlorophenol	Not determined	-
DIORTHOTOLYL GUANIDINE	> 100	Not determined

Validity criteria fulfilled:

yes

Remarks:

see conclusions

Conclusions:

The purpose of this study was to determine the effect of the test item on bacteria according to the OECD guideline 209 "Activated Sludge, Respiration Inhibition Test" and the OECD principles of Good Laboratory Practices.

1,3-di-o-tolylguanidine partly inhibited the respiration of activated sludge in the conditions of the test up at some test concentrations. NOEC was 22.3 mg/L.

The test can be considered as valid because the quality criteria were fulfilled

Executive summary:

The purpose of this study was to determine the effect of the test item on bacteria according to the OECD guideline 209 "Activated Sludge, Respiration Inhibition Test" and the OECD principles of Good Laboratory Practices.

 

The effect of the test item on bacteria is assessed by measuring the micro-organisms respiration rate on a sample of activated sludge during an incubation period of 3 hours.

 

TheEC₅₀calculationsofrespiration inhibitionrate of reference and test substance are presented in the table hereafter:

                                                              EC₅₀- 3h (mgIL)

                                                                  Value                                    95%Cl*

         3,5-Dichlorophenol                              18                                             -

         1,3-di-o-tolylguanidine                     > 100                                         ND

 

*Cl: Confidence Interval, not determined for the test item as EC₅₀ was > 100 mg/L.

 

1,3-di-o-tolylguanidine partly inhibited the respiration of activated sludge in the conditions of the test up at some test concentrations. NOEC was 22.3 mg/L.

 

 

The test can be considered as valid because the quality criteria were fulfilled

Description of key information

3h-EC10DOTG = 22.3 mg/L.
Study performed in accordance with:
- OECD testing guideline 209 and 
- GLP requirements. 

 

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

22.3 mg/L

Additional information

The purpose of this study was to determine the effect of the test item on bacteria according to the OECD guideline 209 "Activated Sludge, Respiration Inhibition Test" and the OECD principles of Good Laboratory Practices.

 

The effect of the test item on bacteria is assessed by measuring the micro-organisms respiration rate on a sample of activated sludge during an incubation period of 3 hours.

 

The EC₅₀ calculations of respiration inhibitionrate of reference and test substance are presented in the table hereafter:

                                                               EC₅₀- 3h (mgIL)

                                                             Value                                    95%Cl*

3,5-Dichlorophenol                             18                                             -

1,3-di-o-tolylguanidine                    > 100                                         ND

 

*Cl: Confidence Interval, not determined for the test item as EC₅₀was > 100 mg/L.

 

1,3-di-o-tolylguanidine did not show any effect on the respiration of activated sludge in the conditions of the test up to a concentration of 100 mg/L.

 

 

The test can be considered as valid because the quality criteria were fulfilled