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Toxicity to microorganisms

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Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Although considered a Klimisch 3, the study is of an adequate quality for the assessment of microbial toxicity as part of a weight-of-evidence in conjunction with other similar endpoints. In accrodance with ECHA Guidance on Information Requirements anc Chemical Safety Assessment, Chapter R.7B, toxicity data on ciliated protozoa can be used for deriving PNECstp.
Principles of method if other than guideline:
Cell multiplication test: Dissolved toxic water ingredients will inhibit cell multiplication of the protozoon, U. parduczi. Thus, in a test culture containing dissolved toxic substances the count of organisms, after a certain period, will be less than in a test culture kept under identical conditions, however free from toxic influence. The number of protozoa is determined by means of a cell counter.
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Uronema parduzci
Details on inoculum:
The inoculum of U. parduczi was in the log phase of growth.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
20 h
Test temperature:
25 °C
Reference substance (positive control):
not specified
Key result
Duration:
20 h
Dose descriptor:
NOEC
Effect conc.:
3 140 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: Cell multiplication
Validity criteria fulfilled:
yes
Conclusions:
In a study on the effects of allylamine on cell multiplication in the ciliated protozoa U. parduczi, the 20 h NOEC was determined to be 3,140 mg/L.
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Although not conducted to GLP and containing deficiencies in the detail of reporting, the data are of an adequate quality for the assessment of microbial toxicity as part of a weight-of-evidence in conjunction with other similar endpoints. Pseudomonas putida is considered one of the more sensitive species for microbial toxicity testing (UBA 1993), and ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7B state that cell multiplication inhibition tests with P. putida may be used for the calculation of PNECmicro-organisms in cases where no other are available.
Qualifier:
equivalent or similar to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Pseudomonas putida
Details on inoculum:
Axenic cultures of P. putida were used. Organisms were in the log phase of growth
Test type:
static
Water media type:
freshwater
Total exposure duration:
6 h
Test temperature:
25 °C
Reference substance (positive control):
not specified
Key result
Duration:
6 h
Dose descriptor:
NOEC
Effect conc.:
700 mg/L
Nominal / measured:
nominal
Basis for effect:
other: Cell multiplications
Validity criteria fulfilled:
yes
Conclusions:
The NOEC of allylamine to P. putida is 700 mg/L.
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Although not conducted to GLP and containing deficiencies in the detail of reporting, the data are of an adequate quality for the assessment of microbial toxicity as part of a weight-of-evidence in conjunction with other similar endpoints. In accordance with ECHA Guidance on Information Requirements anc Chemical Safety Assessment, Chapter R.7B, toxicity data on ciliated protozoa can be used for deriving PNECstp.
Principles of method if other than guideline:
Cell multiplication test: Dissolved toxic water ingredients will inhibit cell multiplication of the protozoon, C. paramecium. Thus, in a test culture containing dissolved toxic substances the count of organisms, after a certain period, will be less than in a test culture kept under identical conditions, however free from toxic influence. The number of protozoa is determined by means of a cell counter.
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Chilomonas paramaecium
Details on inoculum:
The inoculum of C. paramecium was in the log phase of growth.
Test type:
other: Cell multiplication
Total exposure duration:
48 h
Test temperature:
20 °C
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
7.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Cell multiplication
Validity criteria fulfilled:
yes
Conclusions:
In a study on the effects of allylamine on cell multiplication in the ciliated protozoa C. paramecium, the 48 h NOEC was determined to be 7.7 mg/L.
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 May 2013 to 20 June 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study presents an activated sludge respiration inhibition preliminary test conducted in accordance with international guidelines (OECD 209) and in compliance with the applicable GLP Regulations. The full study was not conducted as the preliminary test supported the validity of NOECs from existing data. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Only preliminary study conducted
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The method of preparation used during the range finding test was based on the results of a solubility trial, where concentrations of the test substance in the dilution water at 10 and 1000 mg/L, were analysed, using liquid chromatography with tandem mass spectrometric detection, at 0, 30 and 180 minutes. Recoveries ranged from 93 to 143% and did not decrease with time, indicating that direct addition of the test substance to the dilution water was suitable formulation for the test mixtures.
Aliquots of the test substance (6.55, 65.5 and 655 µL) were added directly to dilution water in the test bottles (500 mL) and made up to volume, to provide test media with nominal concentrations of 10, 100 and 1000 mg/L, respectively.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105 °C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight, at 20 ± 2 ºC.
On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not applicable
Test temperature:
Refer to Table 1.
pH:
Refer to Table 1.
Dissolved oxygen:
Dose-response presented in attached illustration, raw data not presented.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal Concentrations: 0, 10, 100 and 1000 mg/L
Nominal Reference Substance Concentrations: 3, 10 and 32 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-DCP
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
234 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:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The dose-response curves for the test and reference item are presented in the attached illustration.
Results with reference substance (positive control):
The three-hour EC50 for 3,5-DCP (7.18 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 2 to 25 mg/L).

Table 1. Temperature and pH Measurements

   Nominal Concentration    Temperature (°C) pH    
 Group  (mg/L)  Bottle No.  Initial  Final  Initial  Final
 Control  -  1  20.5  20.5  7.15  7.67
     2  20.5  20.6  7.15 7.71 
     3  20.5  20.6  7.15 7.72
     4  20.5  20.5  7.17 7.74
     5  20.7  20.6  7.07 7.62
 Test  10  7  20.8  20.7  7.79  7.87
     8  20.7  20.7  7.38  7.79
     9  20.7  20.6  7.38  7.70
     10  20.7  20.6  7.46  7.87
     11  20.6  20.5  7.36  7.70
 Test  100  13  20.5  20.5  8.54  8.06
     14  20.4  20.5  8.53  8.13
     15  20.5  20.5  8.56  8.03
     16  20.4  20.5  8.51  8.13
     17  20.5  20.5  8.53  8.03
 Test  1000  19  20.6  20.8  10.38  9.89
     20  20.6  20.7  10.38  9.88
     21  20.7  20.8  10.40  10.03
     22  20.6  20.8  10.38  9.85
     23  20.6  20.8  10.41  9.97
 Reference  3  25  20.5  20.8  7.27  7.95
   10  26 20.6  20.9   7.24  7.79
   32  27  20.6  20.9  7.30  7.80

The pH of the samples was considered to be high for the 100 and 1000 mg/L group (outside the specified pH 7±0.5) but investigations indicated that this higher pH did not cause any inhibition of activated sludge under test conditions and as such any inhibition observed would be a result of the test material.

Table 2. Statistical Analysis

 Nominal Concentration (mg/L)  Sample size  Mean Specific Respiration Rate (Rs mgO2.gh)  % Inhibition  p
 Control  5  33.4  0.00  -
 10  5 30.8   7.73  0.095
 100  5  24.1  27.8  <0.001
 1000  5  4.70  85.9  <0.001

p values are for the comparison with the Control using Williams' test (W).

Note negative inhibition values indicate mean specific respiration rates that are greater than the mean control specific respiration rates.

Data has been calculated using unrounded values.

Validity criteria fulfilled:
yes
Conclusions:
Allylamine significantly inhibited the respiration rates of the samples of activated sludge at 100 and 1000 mg/L, and the 50% effect concentration (EC50) for inhibition was calculated to be 234 mg/L (95 % confidence limits of 177 and 310 mg/L).
The no observed effect concentration (NOEC) for allylamine was 10 mg/L.
The three-hour EC50 for 3,5-DCP (7.18 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 2 to 25 mg/L). The validity criterion relating to the respiration rates in the control (specific respiration rate ≥ 20 mgO2/gh and a coefficient of variation of ≤ 30%) was also satisfied.
Executive summary:

The objective of the study was to assess the effect of allylamine on the respiration rate of activated sludge using the methods detailed in OECD Procedure 209 of the “Guidelines for Testing of Chemicals”: Activated Sludge, Respiration Inhibition Test (carbon and ammonium oxidation), adopted 22 July 2010. Samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed to concentrations of the test substance for three hours. Rates of oxygen consumption were determined using oxygen electrodes and were compared with those of five replicate controls that contained activated sludge and synthetic sewage alone. The nominal allylamine concentrations used in the study were 10, 100 and 1000 mg/L; five replicates vessels were prepared for each concentration including the control. Single replicate vessels of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control. Two additional control vessels were prepared and incubated alongside the reference vessels. The mean specific respiration rate (Rs) of the control vessels incubated with the test vessels was 33.4 mgO2/gh with a coefficient of variation (CV) of 6.6%. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 7.18 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. Allylamine significantly inhibited the respiration rates of the samples of activated sludge at 100 and 1000 mg/L, and the 50% effect concentration (EC50) for inhibition was calculated to be 234 mg/L. No definitive phase was conducted at the request of the Sponsor. The no observed effect concentration (NOEC) for allylamine was 10 mg/L.

Description of key information

NOEC = 10 mg/L, activated sludge, OECD 209, Allen (2013)

NOEC = 700 mg/L, P. putida, DIN 38412, part 8 (Pseudomonas Zellvermehrungshemm-Test) , Bringmann & Kuhn (1977)

NOEC = 3,140 mg/L, U. parduczi, Bringmann & Kuhn (1980)

NOEC = 7.7 mg/L, C. paramecium, Bringmann et al., (1980)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
7.7 mg/L

Additional information

For the endpoint "Toxicity to Microorganisms" a weight of evidence approach was used based on four studies; a preliminary activated sludge respiration inhibition test (OECD 209), a toxicity study on the bacterium P. putida, and two studies on ciliated protozoans (U. parduczi and C. paramecium). The preliminary OECD 209 study in support of the toxicity data on the relevant individual microorganism species is suitable for the derivation of PNECSTP.

The lowest available NOEC was used for the chemical safety assessment.