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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 February 2018 - 16 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Test item information
Appearance: Clear light amber liquid
Purity/Composition: >90%
Test item storage: At room temperature

Additional information
Test Facility test item number: 209077/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Glyceryl Triacetyl Ricinoleate
CAS number: 101-34-8
Molecular formula: C63H110O12
Molecular weight: 1059
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Solubility in water: Insoluble
Stability in water: Stable
Analytical monitoring:
yes
Details on sampling:
Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis. The method of analysis is described in the appended Analytical Report (Appendix 5).
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 1.0 mL from the approximate centre of the test vessels
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the limit concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
The batch of Dermol GTR tested was a clear light amber liquid with a purity >90% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure, except for the WAF prepared at 100 mg/L which was colorless and slightly hazy. Immediately after preparation of the test solutions, the WAF prepared at a loading rate of 100 mg/L was checked for the Tyndall effect, using a Laser pen. No scattering of the light bundle was observed which indicates that only the dissolved part of the test item was present in the test solution.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Preliminary Data
The water solubility of Dermol GTR at 20°C was determined to be <1.5 µg/L, using the slow-stirring flask method (Test Facility Study No. 20136813).
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test System
- Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
CaCO3: 24 mg/l
Test temperature:
22 - 24 °C
pH:
8.1 - 8.3
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 1.0, 10.0, & 100.0 mg/l Loading rate
Details on test conditions:
Testing Strategy and Experimental Design
Test Concentrations
- Dermol GTR: WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L.
- Control: Test medium without test item or other additives.
- Replicates: 6 replicates of the control and WAF prepared at 100 mg/L,
3 replicates of WAFs prepared at loading rates of 1.0 and 10 mg/L,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.
Test Procedure and Conditions
- Test duration: 72 hours
- Test type: Static
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: An initial cell density of 1 x 104 cells/mL.
- Illumination: Continuously using TLD-lamps with a light intensity of 83 µE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings
- pH: At the beginning and at the end of the test, for the control and WAF prepared at a loading rate of 100 mg/L.
- Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae compared to the control. 4.9.1.
Recording of Cell Densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and at the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

INTERPRETATION
Acceptability of the Test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 217).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 18%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 0.52%).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Exceeds water solubility
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Exceeds water solubility
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: Exceeds water solubility
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Exceeds water solubility
Details on results:
Measured Test Item Concentrations
Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test were 17 mg/L, which decreased to 0.033% of initial at the end of the test. The limit concentration was observed to be slightly hazy at the start and at the end of the test in all test vessels, and in the test vessel without algae after 48 hours of exposure. A small response was recorded in the control at the end of the test. Contribution was, however, considered negligible. The concentrations in the samples taken from solutions with and without algae were slightly different throughout the entire exposure period, which indicates that the presence of the algae presumably affected the test item concentration in test medium. Based on these results, effect parameters were based on the Time Weighted Average (TWA) of 1.2 mg/L calculated at the limit concentration (see Table 1).
The measured concentrations during the entire test period exceeded the water solubility, determined to be < 1.5 µg/L (Test Facility Study No. 20136813). Hence, it is likely that the over-saturation explained the high decrease of the test item concentration during the test period. It can stated that testing was performed at the maximum soluble concentration of test item in test medium with a TWA concentration of 1.2 mg/L.

Mean Cell Densities
Figure 1 shows growth curves at the control and the limit concentration of Dermol GTR. The individual and group mean cell densities measured at 24h intervals are given in Table 7.

Inhibition of Growth Rate and Inhibition of Yield
Table 2 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 3 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 4 (see Appendix 1 for the individual values).
Growth rate and Yield at the limit concentration were statistically significantly inhibited by 7.7 and 34%, respectively, at the end of the test. For growth rate, however, effects were considered as biologically not relevant (i.e. <10%) and therefore, the NOEC based on biological relevance was set at the TWA concentration of 1.2 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at a loading rate of 100 mg/L when compared to the control.

Determination of Effect Concentrations
Table 5 shows the effect parameters based on the TWA concentration of 1.2 mg/L calculated at the limit concentration.

Experimental Conditions
Table 6 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 22 and 24°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Table 1: Measured Concentrations Versus Loading rate

Dermol GTR WAF-specific loading rate (mg/L) Measured concentration (mg/L) TWA conc. (mg/L)
t=0h t=24h t=72 h
100 16.8 0.764 0.00554 1.2

Table 2: Growth Rate And Percentage Inhibition For The Total Test Period

Dermol GTR Loading rate (mg/L) Mean Std. Dev. n %Inhibition
Control 1.793 0.0094 6  
1 1.797 0.0032 3 -0.19
10 1.773 0.0149 3 1.1
100 (1.2) 1.655 0.0155 6 7.7#

#– effect statistically significant but biologically not relevant (<10%), ( ) – TWA concentration (mg/L).

Table 3: Growth Rate And Percentage Inhibition At Different Time Intervals

Dermol GTR Loading rate (mg/L) n 0 – 24 h 24 – 48 h 48 – 72h
Mean %Inhibition Mean %Inhibition Mean %Inhibition
Control 6 2.09   1.832   1.457  
100 (1.2) 6 2.069 1.014 1.716 6.331 1.181 18.959

( ) – TWA concentration (mg/L).

Table 4: Yield And Percentage Inhibition For The Total Test Period

Dermol GTR Loading rate (mg/L) Mean Std. Dev. n %Inhibition
Control 216 6.05 6  
1 218.1 2.1 3 -0.98
10 203.5 9.14 3 5.8
100 (1.2) 142.6 6.61 6 34*

* – effect statistically significant, ( ) – Time Weighted Average concentration (mg/L).

Table 5: Effect Parameters

Parameter (mg/L) NOEC* NOEC** EC10 EC50
Growth rate <1.2 1.2 >1.2 >1.2
Yield  <1.2 <1.2 <1.2 >1.2

* - based on statistical significance, ** - based on biological relevance.

Table 6: pH Levels Recorded During the Limit Test

Dermol GTR Loading rate (mg/L) pH
t=0h t=72h
Control 8.3 8.3
100 (1.2) 8.1 8.2

( ) – TWA concentration (mg/L).

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the tested concentrations of Dermol GTR.
The EC50 for growth rate inhibition (72h-ERC50) and yield inhibition (72h-EYC50) exceeded the solubility limit of test item in test medium with a TWA concentration of 1.2 mg/L.
The 72h-NOEC for growth rate inhibition was below the TWA concentration of 1.2 mg/L based on statistical significance.
The 72h-NOEC for growth rate inhibition was 1.2 mg/L (TWA concentration) based on biological relevance.
The 72h-NOEC for yield inhibition was below the TWA concentration of 1.2 mg/L based on both statistical significance and biological relevance.
Executive summary:

The objectiveofthe study was to evaluate Dermol GTR for its ability to generate toxic effects inRaphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata)during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Dermol GTR tested was a clear light amber liquid with a purity of >90% which was not completely soluble in test medium at the loading rates initially prepared.Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L in the limit test. In addition, three replicates per group were exposed to WAFs prepared at loading rates of 1.0 and 10 mg Dermol GTR per litre in a combined range-finding test. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed. The measured concentration at the start of the test were 17 mg/L, which decreased to 0.033% of initial at the end of the test. The limit concentration was observed to be slightly hazy at the start and at the end of the test in all test vessels, and in the test vessel without algae after 48 hours of exposure. A small response was recorded in the control at the end of the test. Contribution was, however, considered negligible. Analytical results of the samples taken from solutions with and without algae were slightly different throughout the entire exposure period, which indicates that the presence of the algae presumably affected the test item concentration in test medium. Based on these results, effect parameters were based on the Time Weighted Average (TWA) of 1.2 mg/L calculated at the limit concentration.It should be noted that the water solubility of the test item was determined to be <1.5µg/L (Test Facility Study No. 20136813), which likely explained the high decrease of the concentration during the test period. 

Growth rate and Yield at the limit concentration were statistically significantly inhibited by 7.7 and 34%, respectively, at the end of the test. For growth rate, however, effects were considered as biologically not relevant (i.e. <10%) and therefore, the NOEC based on biological relevance was set at the TWA concentration of 1.2 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on the TWA concentration) obtained in this study are summarized in the table below.

Parameter (mg/L)

NOEC*

NOEC**

EC10

EC50

Growth rate

<1.2

1.2

>1.2

>1.2

Yield

<1.2

<1.2

<1.2

>1.2

* - based on statistical significance, ** - based on biological relevance.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Additional information

EC50 exceeds water solubility of the registered substance.