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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:
7 January 2013 - 1 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item without emulsifier was investigated.

Source:
Amidoamine 2 (UVCB, based on hydrogenated tallow instead of hydrogenated palm oil):
- Name of test material (as cited in study report): FCM (DETA)M C16-18 TLW hydrogenated (Amidoamine 2 (UVCB) based on hydrogenated tallow instead of hydrogenated palm oil)
- Substance type: Amidoamine
- Chemical name: Amides, from diethylenetriamine and hydrogenated tallow / Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
- CAS 68920-82-1
- Physical state: pale yellowish solid at 20 °C
- Batch No.: PU22340011
- Expiry date of batch: 21 August 2014
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Since the test item is a multi-component substance that is only partially soluble, the water accommodated fraction (WAF) approach was used. The loading of the test material was prepared by direct addition of the respective amount of test item to the aerated algal medium, moderate stirring for 96 h, followed by filtration (0.45 µm cellulose acetate membrane sterile syringe filter, VWR International). The resulting water soluble fraction (WSF) was used in the test.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Axenic slope culture of Desmodesmus subspicatus 86.81 SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
OECD algal medium
Test temperature:
22 ± 2 °C, thermo-controlled room
pH:
The pH-value in the WAF replicates decreased by 0.2 units from 8.1 to 7.9.
Nominal and measured concentrations:
Analytical determinations of the test item concentration were based on dissolved organic carbon (DOC). Determinations were conducted at the beginning,
after 24, 48 and 72 h of exposure. Net DOC concentrations of the test item were below quantification limit (LOQ=0.5 mg C/l). Thus, the evaluation was based on the loading rate.
Details on test conditions:
As in the range-finding test 250 mL glass flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group. The control group was maintained under identical conditions but not exposed to the test item. An exponentially growing preculture of algae was used to inoculate sterile test flasks characterized by 0.50–0.85 µg/ml with respect to dry weight corresponding to about 2–5•10^3 cells/ml (i.e. OD680 of about 0.005 units). Test flasks were placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
Cell concentrations were determined every 24 h using a spectrophotometer (680 nm wavelength). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the control vessels.
Illumination was continuously sourced from a Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland).Light intensity amounts to about 3000 lux which corresponds to about 40 μE•m-2•s-2 according to the OECD TG no. 2013. The light intensity is maintained within ±15% from the average light intensity over the incubation area.
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
Results with reference substance (positive control):
The culture is yearly re-purchased. Potassium dichromate is used to control the sensitivity of the culture twice a year. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
The statistical significance of differences between effects observed for control and test substance treatment was determined by Dunnett’s test using the ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany).
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

In the Klimisch 1 GLP study from Caduff (2013) the effect of the test item on the growth of Desmodesmus subspicatus was determined in a 72‑hour static limit test according to Part C.3 of the Commission Regulation (EC) No 440/2008 and the OECD Guideline for Testing of Chemicals No. 201 (2006). Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). The nominal test item concentration tested was 100.0 mg/L. Additionally, a control group was tested in parallel. The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
8 October 2015 - 28 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An OECD 201GLP study for a similar substance is available and is used within a read-across approach. The composition of source and target substance is described and discussed in detail in the attached supporting information as well as the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Target substance:

Amidoamine 2 (UVCB, low nitrogen):
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
CAS No.: 1618093-67-6
New chemical name: Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: PU50070067
Purity: 100 % (UVCB)
Storage condition of test material: Room temperature, protected from light
Stability: stable under test conditions
Analytical monitoring:
yes
Details on sampling:
- Concentrations: WAF of 100 mg/l test item
- A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
Vehicle:
no
Details on test solutions:
The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate
such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under
constant aeration and illumination at 21 ± 1 °C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
NaHCO3 15.0 mg/L culture medium
CaCl2.2H2O 4.41mg/L culture medium
Test temperature:
24 ± 1 ºC, thermo-controlled room
pH:
7.5
Nominal and measured concentrations:
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for chemical analysis. It is evident from this work that increasing the stirring period did not increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
Details on test conditions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.96 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 7.2 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
Chemical analysis of the test preparations at 0 and 72 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0047 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Given that toxicity cannot be attributed to a single component or mixture of components but to
the test item as a whole, the results were based on nominal loading rates only.
Results with reference substance (positive control):
A positive control (Study Number 41501180) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.0 mg/L; 95% confidence limits 0.90 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.49 mg/L; 95% confidence limits 0.42 – 0.58 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It is considered likely, that the target substance Amidoamine 2 (UVCB, low nitrogen) will show a comparable toxicity to aquatic algae and cyanobacteria as Amidoamine (UVCB) based on the similar chemical composition.
Executive summary:

In the Klimisch 1 GLP study from Vryenhoef (2015) the effect of the test item on the growth of Pseudokirchneriella subcapitata was determined in a 72‑hour static limit test according to Part C.3 of the Commission Regulation (EC) No 440/2008


and the OECD Guideline for Testing of Chemicals No. 201 (2006). Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). The nominal test item concentration tested was 100.0 mg/L. Additionally, a control group was tested in parallel. The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.


It is considered likely, that the target substance Amidoamine 2 (UVCB, low nitrogen) will show a comparable toxicity to aquatic algae and cyanobacteria as Amidoamine (UVCB) based on the similar chemical composition. It is expected that the higher concentration of saturated mono- and diglycerides and the presence of saturated triglycerides in Amidoamine 2 (UVCB, low nitrogen) will have no adverse effect on aquatic algae and cyanobacteria.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 October 2015 - 28 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item without emulsifier was investigated.

Source substance:

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
Former CAS No.: 1618093-67-6
New chemical name: Triglycerides, C16-18 (even-numbered), reaction products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2018
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target substance:

Amidoamine 2 (UVCB, low nitrogen):
Former chemical name: Amides, from diethylenetriamine and hydrogenated palm oil
CAS No.: 1618093-67-6
New chemical name: Glycerides, C16-18 (even numbered) and their amidation products with diethylenetriamine
Physical state: pale yellowish solid at 20 °C
Batch No.: PU50070067
Purity: 100 % (UVCB)
Storage condition of test material: Room temperature, protected from light
Stability: stable under test conditions
Analytical monitoring:
yes
Details on sampling:
- Concentrations: WAF of 100 mg/l test item
- A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
Vehicle:
no
Details on test solutions:
The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate
such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under
constant aeration and illumination at 21 ± 1 °C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
NaHCO3 15.0 mg/L culture medium
CaCl2.2H2O 4.41mg/L culture medium
Test temperature:
24 ± 1 ºC, thermo-controlled room
pH:
7.5
Nominal and measured concentrations:
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for chemical analysis. It is evident from this work that increasing the stirring period did not increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
Details on test conditions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.96 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 7.2 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
Chemical analysis of the test preparations at 0 and 72 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0047 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Given that toxicity cannot be attributed to a single component or mixture of components but to
the test item as a whole, the results were based on nominal loading rates only.
Results with reference substance (positive control):
A positive control (Study Number 41501180) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.0 mg/L; 95% confidence limits 0.90 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.49 mg/L; 95% confidence limits 0.42 – 0.58 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

In the Klimisch 1 GLP study from Vryenhoef (2015) the effect of the test item on the growth of Pseudokirchneriella subcapitata was determined in a 72‑hour static limit test according to Part C.3 of the Commission Regulation (EC) No 440/2008


and the OECD Guideline for Testing of Chemicals No. 201 (2006). Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). The nominal test item concentration tested was 100.0 mg/L. Additionally, a control group was tested in parallel. The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.


It is considered likely, that the target substance Amidoamine 2 (UVCB, low nitrogen) will show a comparable toxicity to aquatic algae and cyanobacteria as Amidoamine (UVCB) based on the similar chemical composition. It is expected that the higher concentration of saturated mono- and diglycerides and the presence of saturated triglycerides in Amidoamine 2 (UVCB, low nitrogen) will have no adverse effect on aquatic algae and cyanobacteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information