Quaternary ammonium compounds, bis(C16-18 and C18-unsatd. alkyl)dimethyl, chlorides

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

received for publication: December 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Nitrification inhibition was assessed using Rhine water (naturally contained nitrifying microorganisms). Test duration: 48 hours

GLP compliance:

no

Analytical monitoring:

no

Remarks:

not for test item, but for ammonia

Details on sampling:

Samples were taken, filtered over a 0.22 µm filter and stored at -80 °C.

Vehicle:

no

Details on test solutions:

Stock solutions (freshly prepared before use):
An aliquot of DTDMAC was liquified in a water bath (50 °C) and homogenized prior to weighing.
Dilution with dilution water, stirred for approximately 15 minutes.
Appearance of stock solutions: uniformly opaque, whitish.

Preconditioning of glass ware (adsorptive properties of test item):
All glassware was rinsed with 99% chloroform and 99% methanol before use.
During the experiments the glassware was rinsed with demineralized water only.

Dilution water used for prepartion of concentration levels and control media:
Water from the river Rhine which was sampled in autumn 1989: suspended solids concentration 45 mg/L; DOC 4.0 mg/L.

Test organisms (species):

other: natural Rhine water including nitrifying micro-organisms

Details on inoculum:

Water from the river Rhine which was sampled in autumn 1989: suspended solids concentration 45 mg/L; DOC 4.0 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

252 mg/L CaCO3

Test temperature:

20 °C

pH:

7.7

Dissolved oxygen:

not reported

Nominal and measured concentrations:

See IUCLID section "Any other information on results incl. tables" below.

Details on test conditions:

Inoculum used: water from the river Rhine, naturally containing nitrifying bacteria; water sampled in autumn 1989; suspended solids concentration: 45 mg/L; DOC: 4.0 mg/L.
The experiment with nitrifying bacteria was carried out after acclimatization to 20 °C (circa 24 hours) and adjustment of the natural ammonium concentration for Rhine water.
Tests were performed in continuously stirred batches in the dark.
Samples were taken, filtered over a 0.22 µm filter and stored at -80 °C. The ammonium concentrations were determined with a Technicon Autoanalyzer II system.
Endpoint: ammonium oxidation;
Temperatue: 20 °C;
Replicates: one experiment, 2 replicates.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.36 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of nitrification rate

Remarks:

assessed via ammonium depletion

Remarks on result:

other: using Rhine river water (suspended solids concentration 45 mg/L; DOC 4.0 mg/L) as the inoculum

Details on results:

See IUCLID section "Any other information on results incl. tables" below.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

NOEC-values were derived from the observed concentration-effect relationship. However, in case of graded responses the Dunnett's test was carried out to demonstrate significant differences between the control group and the exposed groups. The Toxstat-program version 2.1 was used for these statistical tests (Gulley et al., 1988).

Details on results:

	Ammonium oxidation, results as deduced from Figure 1 of the publication
concentration test material (mg/L)	Nominal active substance concentr. (78.7% of product) [mg/L]	% of control
Control	Control	100
0.03	0.0236	114
0.1	0.0787	115
0.3	0.2361	132
1	0.7870	85
3	2.3610	90
10	7.8700	-36

At the lower concentrations of the test item, a slightly stimulating effect was observed. At the highest test item concentration used (7.87 mg/L active ingredient), -36% was observed in comparison to the control. This corresponds to an increase in ammonium compared to the ammonium concentration at the start. This possibly could be due to a release of ammonium from the test item by the action of carbon oxidizing microorganisms and inhibition of nitrifying bacteria at the same time, such that an accumulation of ammonium occurred.

Based on the data shown above, the authors report a NOEC (test material) of 3 mg/L.

Excluding the solvent part of the test item, a relevant NOEC based on active ingredient content (78.7% active ingredient) of 2.36 mg/L is the relevant value for nitrification inhibition determined with natural Rhine water via the decline of ammonium.

Validity criteria fulfilled:

not applicable

Conclusions:

Nitrification inhibition (ammonium oxidation) using natural river water (Rhine river; Roghair et al, 1992):
NOEC (Rhine river water; 48 h; nitrification inhibition via ammonium oxidation; active ingredient) = 2.36 mg/L (nominal concentration)

Executive summary:

DHTDMAC was tested for toxicity towards nitrifying bacteria in a 48 h test on ammonium oxidation using natural Rhine river water (suspended solids concentration 45 mg/L; DOC 4.0 mg/L) as the inoculum (Roghair et al., 1992). The following results were determined:

At the lower concentrations of the test item, a slightly stimulating effect was observed. At the highest test item concentration used (7.87 mg/L active ingredient), -36% was observed in comparison to the control. This corresponds to an increase in ammonium compared to the ammonium concentration at the start. This possibly could be due to a release of ammonium from the test item by the action of carbon oxidizing microorganisms and inhibition of nitrifying bacteria at the same time, such that an accumulation of ammonium occurred.

Based on these data, a relevant NOEC based on active ingredient content of the test material used (78.7% active ingredient) of 2.36 mg/L is the relevant value for nitrification inhibition determined with natural Rhine water via the decline of ammonium.

Description of key information

Nitrification inhibition (ammonium oxidation) using natural river water (Rhine river; Roghair et al, 1992):

NOEC (Rhine river water; 48 h; nitrification inhibition via ammonium oxidation; active ingredient) =  2.36 mg/L (nominal concentration);

In spite of this reliable (RL2) and relevant (nitrification inhibition) data, for consistency reasons the value used in the EU Risk Assessment Report on DODMAC (2009) was used as key value (see additional information below).

Key value for chemical safety assessment

EC50 for microorganisms:

2.1 mg/L

Additional information

Read across for DTDMAC from structurally similar Quats DHTDMAC/DODMAC can be applied and the corresponding effect data form DHTDMAC/DODMAC used for DTDMAC as well.

Below, an Overview table with the available toxicity tests to microorganisms is given.

Summary table microorganism toxicity - relevant available data

Inoculum	Endpoint	(No)Effect Conc.	Substance	Reference
Pseudomonas putida	18h EC50	48 / 58 mg/l	DHTDMAC	UBA, 1992
secondary effluent	5d EC50	2.0 / 6.5 mg/l	DHTDMAC	UBA, 1992
activated sludge	3h EC50	520 mg/l	DHTDMAC	UBA, 1992
activated sludge	3h EC50	278 mg/L	DHTDMAC	Noack, 1993 (OECD 209)
nitrifying bacteria (contained in Rhine river water, susp. solids: 45 mg/L; DOC 4.0 mg/L)	48 h NOEC	2.36 mg/L	DHTDMAC	Roghair et al., 1992
nitrifying bacteria	>119h IC50	2.1 mg/l	DHTDMAC	Wagner & Kayser,1990
Aerobic bacteria	3h EC20 3h EC50 3h EC80	131 mg/l 278 mg/l 590 mg/l	DHTDMAC	Hoechst, 1993
Anaerobic bacteria	28d NOEC	≥200 mg/L	DTDMAC	Akzo, 1990

 

Except for the data given above from Noack (1993), Roghair et al. (1992), and Akzo (1990), the data were already considered in the EU Risk Assessment Report on DODMAC (2009). These yet unconsidered data do allow for the following conclusions:

• Up to 200 mg/L DHTDMAC no inhibition of gas production could be observed testing anaerobic sludge consortia (Akzo, 1990). This is an important insight with regard to sludge digestion.
• Noack (1993) essentially confirms the low toxicity of DHTDMAC towards total respiration assessed in an activated sludge respiration inhibition test (OECD 209).
• The data by Roghair et al. (1992) included do confirm the special relevance of nitrifying bacteria: nitrification inhibition (ammonium oxidation) was assessed using natural river water (Rhine river; Roghair et al, 1992): NOEC (Rhine river water; 48 h; nitrification inhibition via ammonium oxidation; active ingredient) =  2.36 mg/L (nominal concentration).

The reliable and relevant data according to Roghair et al. based on natural river water actually are deemed to be the most relevant for hazard and risk assessment. Compared to the EU risk assessment, a higher PNEC for STP microorganisms would result (AF 1 for NOEC, PNEC = 2.36 mg/L). However, for the sake of consistency the value used in the EU Risk Assessment Report on DODMAC (2009) based on results by Wagner & Kayser (1990) was used as key value.

From the EU Risk Assessment DODMAC (EU, 2002) the following discussion on Microorganism toxicity is available:

Toxicity to Microorganisms

There are several tests concerning toxicity of DHTDMAC to bacteria which can be used for the derivation of a PNECWWTP, but no test was conducted with DODMAC. In each case labo­ratory water was used.

The toxicity of DHTDMAC to Pseudomonas putidawas investigated in a growth inhibition test according to a GermanDIN-guideline (Bringmann & Kühn method; UBA, 1992). In two tests EC50-values of 48 and 58 mg/l were derived after 18 hours (nominal values, graphically extrapolated).

Secondary effluent of a domestic waste water treatment plant was used as inoculum in a closed-bottle inhibition test (OECD 301D, UBA, 1992). The graphically extra­polated EC50-values of two tests were 2.0 and 6.5 mg/l (nominal concentrations) after a test duration of 5 days.

In an activated sludge respiration inhibition test (OECD 209) inoculum from a predominantly domestic waste water treatment plant was used (UBA, 1992). A 3h EC50 of 520 mg/l was derived graphically from the dose response curve. The corresponding statistically derived value was 267 mg/l (nominal concentrations).

The toxicity of DHTDMAC to nitrifying bacteria enriched in a laboratory waste water treat­ment plant (domestic sludge originally) was investigated in a manometric respirometer test (Wagner & Kayser, 1990). The test duration in the reference was referred to between 119 and 254 hours for different substances and was stopped when the nitrification of the controls was completed. The IC50 for inhibition of respiration was 2.1 mg/l active ingredient of DHTDMAC (a carrier solvent was used).

Aerobic bacteria from a domestic waste water treatment plant were exposed to DHTDMAC in an OECD 209 test (Hoechst, 1993). The inhibition of respiration was measured after 3 hours and the EC20 was 131 mg/l, the EC50 = 278 mg/l.

Using different safety factors according to the sensitivities of the test systems and the mean effect values the lowest PNEC-values are as follows:

 

Pseudomonas putida     EC50 = 53 mg/l, SF = 100              PNEC = 0.53 mg/l

nitrifying bacteria          EC50= 2.1 mg/l, SF = 10                 PNEC = 0.21 mg/l

secondary effluent          EC50= 4.3 mg/l, SF = 100               PNEC = 0.043 mg/l

 

With all these PNECs it has to be considered that the microorganism toxicity derived in laboratory water tests has to be handled with care as a high influence of the composition of the waste water (e.g. suspended particles, complexing agents) can be assumed, which is the same phenomenon as in surface water tests. Moreover the lowest PNECmicro-organisms of 0.043 mg/l seems to be unrealistic as it is reported that waste water treatment plants operate at DHTDMAC concentrations of 3 to 8 mg/l (chap. 3.1.2.1). However, it is not documented whether the treatment process would be more effective without this DHTDMAC load in the influent and how less adapted plants might react. Nitrifying bacteria were found to be the most sensitive micro-organisms with the lowest EC50 of 2.1 mg/l on which the risk assessment should be based (PNECmicroorganism = 0.21 mg/l) to ensure that the most sensitive treat­ment process can take place.