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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study. However, potential test substance stability issue.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the
control.

For sampling at the end of the test, the test medium of the treatment replicates was pooled.

All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.

The concentrations of the test item RL277/09 were determined in the duplicate test medium samples from the nominal test concentrations of 0.25 to 2.5 mg/L. The samples from the nominal test concentrations of 0.054 and 0.116 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
Details on test solutions:
Test water:
Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.

Dosage: A stock solution of the nominal concentration of 25 mg/L was prepared by dissolving 25.39 mg of the test item completely in 1016 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The stock solution was diluted with test
water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain: No. 61.81 SAG
Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany).

The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.
An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in February 2009 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.1 mg/L (study C36893), range of the 72-hour EC50 for the growth rate from 2000 to 2009: 0.71-1.74 mg/L).
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Hardness:
The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
The test flasks were incubated in a temperature-controlled water bath at a temperature between 21 and 24 °C.
pH:
At the start of the test, the pH measured in the treatments was between 8.3 and 8.5. At the end of the test, pH values of 8.0 to 8.9 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test.
Nominal and measured concentrations:
The selection of the test concentrations was based on the results of range-finding tests (non-GLP).

The nominal concentrations of the test item of 0.054, 0.116, 0.25, 0.54, 1.16 and 2.5 mg/L (corresponding to 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg dry matter/L) were tested in parallel with a control.

The measured concentrations of the test item in the test media of the nominal test concentrations of 0.25 to 2.5 mg/L were between 102 and 110 % of the nominal values at the start of the test.
Details on test conditions:
Lighting: illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measuredlight intensity at the level of the test solutions was approximately 7000 Lux (range: 6510 to 7490 Lux, measured at nine places in the experimental area). The light intensity over the incubation area (measured at nine places in the experimental area) was within ±15% from the average light intensity as recommended by the guideline

Additionally, a control was tested in parallel (test water without test item).

The test design included three replicates per test concentration and six replicates of the control.
The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM).
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.097 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 0.086 – 0.111
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.046 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval 0.045 – 0.048
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.51 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 0.50 – 0.52
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.3
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval 0.29 – 0.31
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.016 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.032 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate

During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 2 to 11% of the nominal values were found. The testing lab suggested that the losses could be due to photolysis of the test item.

Control:

In the control the biomass increased by a factor of 140 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control during 72 hours was 14%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50 is 0.097 mg active substance/L based upon growth rate.
The 72-hour NOEC was determined to be 0.016 mg active substance/L, based on growth.


During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 2 to 11% of the nominal values were found. The testing lab suggested that the losses could be due to photolysis of the test item.
Executive summary:

In a 72-h acute toxicity study, algae (Pseudokirchnerella subcapitata) were exposed to the substance at nominal concentrations of control, 0.054, 0.116, 0.25, 0.54, 1.16 and 2.5 mg test material/L and measured concentrations of 0.016, 0.032, 0.077, and 0.351 mg active substance/L under static conditions. The lowest two test concentrations were not measured analytically. The 72-h EC50was 0.097 mg active substance/L, based on growth. The 72 -hr NOEC was 0.016 mg active substance/L, based on growth.  

This toxicity study is classified as acceptable and satisfies the guideline requirement for toxicity study for algae.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study results generated by reliable QSAR model.
Justification for type of information:
QSAR prediction
Qualifier:
no guideline followed
Principles of method if other than guideline:
The QSAR model, Ecological Structure Activity Relationships (ECOSAR) Class Program to estimate long-term toxicity to algae.
GLP compliance:
no
Analytical monitoring:
not required
Vehicle:
no
Test organisms (species):
other: Green algae
Test type:
other: QSAR
Limit test:
no
Reference substance (positive control):
not required
Dose descriptor:
other: Chronic value
Effect conc.:
8 mg/L
Nominal / measured:
estimated
Conc. based on:
other: QSAR
Remarks on result:
other: Amide structure
Dose descriptor:
other: Chronic value
Effect conc.:
135 mg/L
Nominal / measured:
estimated
Conc. based on:
other: QSAR
Remarks on result:
other: Value for Neutral Tertiary Amine

Acute toxicity data are available for the substance, although long-term toxicity data are unavailable. Instead the registrant has undertaken QSAR modelling using the US EPA’s ECOSAR model in order to generate long-term toxicity data for the substance.

ECOSAR deals with cationic surfactants including quaternary ammoniums as a special case.  The QSARs for cationic surfactants are linear and the ecotoxicity potential is related to the size of the hydrophobic component (i.e. number of carbons – the QSAR are different where carbon chain is greater than C16, or less than C16).  However, the model cannot specifically account for the amide group in the substance molecule (or the double bond at the end of the alkyl chain).  Furthermore, the QSAR for quaternary ammoniums only provides acute toxicity data and these acute toxicity value are low (ca. 1-2 mg/l) for quaternary ammoniums with an average carbon chain of 14. These estimated values are much lower than the measured laboratory values.  It may be reasonable, given the structure of the molecule, to model a lower average carbon chain for the substance, but there is no guidance or accepted experience for using ECOSAR for this type of situation. Furthermore, significant hydrolysis of the amide seems unlikely at relatively neutral pH in the environment.

The ECOSAR operation manual states that “quaternary ammonium compounds (four carbons are bound to the nitrogen) are an exception [to organic ammonium salts] since reduction (e.g., loss of hydrogens) at neutral pH is unlikely; however, these are classified as surfactants.” Instead, the neutral quaternary ammonium compound with four alkyl groups on the nitrogen has been modelled using the ECOSAR programme, but not as a ‘special class’ surfactant due to the reasons described above. However, the compound is unlikely to form the corresponding amine (i.e. loss of an alkyl group) under normal environmental conditions.

Using an ECOSAR approach based on modelling the quaternary ammonium ion provides chronic toxicity data for three trophic levels (Table 1). The SMILES notation used in the assessment for Rewocid 185 UTM is C (=O) (CCCCCCCCC=C) NCCCN{+} (C) (C) C. The most conservative chronic value (equivalent to the geometric mean of a NOEC and LOEC) is for fish (0.5 mg/l).  It can be assumed that the ECOSAR amide predictions provide a worst case estimate for the long-term toxicity of the substance to aquatic organisms; in particular fish appear to be the most sensitive organisms to both the substance and its analogue substance methyl trimethyl-3-[(1-oxododecyl)amino]propylammonium sulphate. The most sensitive organisms in both lab and modelled data are algae for acute toxicity. The algae are several orders of magnitude more sensitive than fish and invertebrates. However, the chronic data indicate much more similar sensitivities across the three trophic levels and indicate fish are the most sensitive.  It is not clear why the sensitivities vary so dramatically in the model and the trend changes between acute and chronic data sets. This presents an uncertainty with the ECOSAR approach.

  

Table 1: ECOSAR chronic aquatic toxicity data (rounded values)

 

 

 

CAS 94313-91-4

CAS 10595-49-0

Organism

Toxicity Test

Value for Amide (mg/l)

Value for Neutral Tertiary Amine (mg/l)

Value for Amide (mg/l)

Value for Neutral Tertiary Amine (mg/l)

Fish

Chronic

0.5

236

0.3

73

Invertebrate

Chronic

29

101

13

36

Algae

Chronic

8

135

5

60

 

In addition, the methylsulphonate coutner ion (SMILES notation CS(=O)(=O)O) was evaluated in ECOSAR and the chronic value for algae was 38,323 mg/l. The modelled results indicate that the amide component of the substanc's structure drives the assessment.

Validity criteria fulfilled:
not applicable
Conclusions:
A chronic value of 8 mg/l relating to the amide structure is provided from modeled results for long-term toxicity of the substance to algae.
Executive summary:

A QSAR-modelled approach using the US EPA's ECOSAR model was used to derive chronic toxicity data for algae based on the amide and neutral tertiary amine components of the test substance structure. The most sensitive chronic value of 8 mg/l was derived for green algae based on the amide structure.

Description of key information

Elementis Specialities (2010) reports a 72-hr LC50 for toxicity in Pseudokirchnerella subcapitata of 0.097 mg active substance/L and a 72-hr NOEC of 0.016 mg active substance/L, based on growth. QSAR modelling using ECOSAR resulted in chronic values for green algae of 8 mg/l and 135 mg/l based on the amide and neutral tertiary amine structures of the substance.

A reliable (Klimisch 2) toxicity study with algae has been conducted on the substance and is included in this dossier. Elementis Specialities (2010) conducted a reliable (Klimisch 2) GLP compliant study following OECD 201 methods with the substance. The algae, Pseudokirchnerella subcapitata, were exposed to the test substance at nominal concentrations of 0.054, 0.116, 0.25, 0.54, 1.16 and 2.5 mg test material/L and measured concentrations of 0.016, 0.032, 0.077, and 0.351 mg active substance/L for 72 hrs under static conditions. The lowest two test concentrations were not confirmed analytically. The 72-h EC50 was 0.097 mg active substance/L, based on growth. The 72-hr NOEC was 0.016 mg active substance/L, based on growth.

 

A QSAR-modelled approach using the US EPA's ECOSAR model was used to derive chronic toxicity data for green algae based on the amide and neutral tertiary amine components of the test substance structure. The most sensitive chronic value of 8 mg/l was derived for green algae based on the amide structure. A chronic value of 135 mg/l was derived for the neutral tertiary amine structure.

 

The measured NOEC for P. subcapitata of 0.016 mg active substance/l is lower than the modelled chronic value and recommended as the basis of PNEC development.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.097 mg/L
EC10 or NOEC for freshwater algae:
0.016 mg/L

Additional information