Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-07-2002 to 12-07-2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
31-07-1992
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name: teflubenzuron
- CAS no.: 83121-18-0
Analytical monitoring:
yes
Details on sampling:
Samples of the test solutions were taken at the beginning and the end of the test period and were stored frozen until analysis by HPLC/UV.
Vehicle:
yes
Remarks:
Acetone
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Two stock solutions were prepared by dissolving the test item in acetone. The test solutions were established by adding 0.1 mL stock solution or acetone to 1,000 mL algal medium. The used medium is recommended by “Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Unversität Göttingen (SAG) – List of Strains” (Schlösser 1982) and had a pH of approximately 6.0.
- Controls: Negative control (algal medium only), solvent control (acetone)
- Evidence of undissolved material: no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: SAG 86.81
- Source: Institut für Pflanzenphysiologie, University Göttingen, Germany
- Age of inoculum: inoculum taken from three days old preculture
- Method of cultivation: according to applied guidelines
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
not reported
Test temperature:
23.39 +/- 0.66°C
pH:
5.9 to 6.5
Dissolved oxygen:
not reported
Salinity:
not applicable
Conductivity:
not reported
Nominal and measured concentrations:
Nominal: 0, 0.02 mg a.s./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: not further specified
- Material, size, headspace, fill volume: glass, 300 mL nominal volume, 100 mL filll volume
- Aeration: algae were kept in suspension by constant shaking at 100 ± 5 oscillations/min
- Initial cells density: 1 x 10e4 cells/mL
- Mean biomass increasing factor for control: 79.8 (negative control) and 90.0 (solvent control), confirming exponential growth
- No. of vessels per concentration: 6
- No. of vessels per control: 6
- No. of vessels per positive control: not specified. A study with the reference item was completed in July 2002.

GROWTH MEDIUM
- Standard medium used: According to the recommendation of the “Sammlung on Algenkulturen”, Pflanzenphysiologisches Institut of the Universtiy Göttingen, Germany (Schlösser, 1982), wich corresponds to the algal mediaum according OECD 201.


OTHER TEST CONDITIONS
- Sterile test conditions: yes (filter-sterilized)
- pH adjustment: no
- Photoperiod: conitnous illumination
- Light intensity and quality: cool white-type fluorescent lamps (mean value of six measurements: 8182 lux)

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: sampled daily and determined using counting chamber and spectrophotometer
Reference substance (positive control):
yes
Remarks:
A seperate study with potassium dichromate was perfored in July 2002.
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.02 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
>= 0.02 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: 72 h 0.996 mg/L, 96 h 1.218 mg/L (biomass)
-
Reported statistics and error estimates:
One-way Analysis of Variance (ANOVA) was used to calculate whether there were significant differences between the growth of algae in the controls and in the test item concentrations.

In the limit test, the acetone stock solution showed a recovery rate of 97.7%. At the beginning of the test, a total recovery of 81.5% of the nominal concentration was measured in the test solution. At the end of the test (96 h), no test item was detected in the test solution with algae, and 32.3% was found in the stability sample without algae. It is assumed that by the end of the test most of the test item was probably either adsorbed or metabolised by the algal cells present in the test solution.


No significant inhibition of growth rate, biomass integral (A) and cell number, com­pared to the control, was observed. Therefore, based on nominal concentrations, a NOEC of ≥ 0.02 mg a.s./L was calculated for the test period 0-72 h and 0-96 h.


 


Table 1: Cell number of Desmodesmus subspicatus dependent on concentration of the test item and time











































































































































































































































































Time



Treatment [mg/L]



Control



Solvent Control



0.020



0h



1.0



1.0



1.0



 



1.0



1.0



1.0



 



1.0



1.0



1.0



 



1.0



1.0



1.0



 



1.0



1.0



1.0



 



1.0



1.0



1.0



Mean:



1.0



1.0



1.0



Std.Dev.:



0.00



0.00



0.00



24 h



3.5



5.0



0.5



 



3.5



2.5



4.0



 



4.5



4.5



3.0



 



3.5



4.5



3.5



 



2.5



4.0



3.5



 



4.5



2.5



5.5



Mean:



3.7



3.8



3.3



Std.Dev.:



0.75



1.08



1.63



48 h



27.5



22.0



23.0



 



20.5



23.5



24.0



 



26.5



23.0



26.0



 



16.5



19.0



13.0



 



24.0



21.0



21.5



 



23.5



21.5



33.5



Mean:



23.1



21.7



23.5



Std.Dev.:



4.05



1.60



6.65



72 h



65.0



113.0



87.5



 



81.0



87.5



93.5



 



95.5



114.5



119.0



 



69.5



75.5



81.0



 



85.5



79.0



107.0



 



82.0



70.5



109.0



Mean:



79.8



90.0



99.5



Std.Dev.:



11.05



19.22



14.48



96 h



159.5



222.5



221.0



 



179.0



178.0



181.5



 



206.0



179.5



217.0



 



172.0



169.0



197.0



 



199.5



122.0



216.0



 



183.0



128.5



228.5



Mean:



183.2



166.6



210.2



Std.Dev.:



17.26



37.06



17.49



n:



6



6



6



CV:



9.4



22.2



8.3


Validity criteria fulfilled:
yes

Description of key information

In a 96 h static toxicity study on the growth of the green algae Desmodesmus subspicatus according to EEC Directive 92/69 method C.3 (1992), the EC50 and the NOEC of the test item were determined to be ≥ 0.02 mg teflubenzuron/L based on nominal concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.02 mg/L

Additional information

One valid toxicity study on the growth of the green algae with the test item according to EEC Directive 92/69 method C.3 (1992) is available. Supportive information from toxicity studies on the growth of the green algae with the metabolites are summarized below, but not documented in separate IUCLID endpoint study records.


All presented studies were peer-reviewed during the assessment of teflubenzuron according Council Directive 91/414/EEC.


 


Key information


ECT (2003): A Study on Toxicity of BAS309 I (teflubenzuron) to Algae (Desmodesmus subspicatus) according to EEC Direc­tive 92/69 method C.3., “Algal Inhibition Test”. Unpublished report,report No. G3AG, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference:  MCA 8.2.6/01


To assess the acute toxicity of the test item to green algae a 96-hour static test with Desmodesmus subspicatus was carried out according to EEC Directive 92/69 method C.3 (1992) in accordance with the GLP principles. The organisms were exposed to a control, a solvent control (0.1 mL acetone/L) and 0.02 mg/L nominal test item concentration. Two stock solutions were pre­pared by dissolving the test item in acetone. The test solutions were established by adding 0.1 mL stock solution or acetone to 1,000 mL algal medium. The used medium is recommended by “Sammlung von Algenkulturen, Pflanzenphysiolo­gisches Institut der Unversität Göttingen (SAG) – List of Strains” (Schlösser 1982) and had a pH of approximately 6.0. The pre-cultures used in the test were 3 days old. The culture conditions were the same as the conditions during the test period. The test vessels were 300 mL Erlenmeyer flasks containing 100 mL test solution. After temperature adaptation, the algal cells were transferred into the test solutions to achieve an initial concentration of 104 cells per mL. The number of replicates in the test item concentration, the control and the solvent control was 6. One addi­tional replicate of the highest test item concentration without algae was prepared to control the stability of the test item under the test conditions. The test vessels were placed onto a shaker (100 ± 5 oscillations/min) under light (continuous illumination at 8,128 lx on average) and temperature (23 ± 2°C) controlled conditions. Samples of the test solutions were taken at the beginning and the end of the test period and were stored frozen until analysis by HPLC/UV. One-way Analysis of Variance (ANOVA) was used to calculate whether there were significant differences between the growth of algae in the controls and in the test item concentrations.


The pH values during the test period were 5.9/6.5 min/max. The temperature was 23.39 ± 0.66°C (n = 241). All test vessels fulfilled the validity criterion for control growth. In the limit test, the acetone stock solution showed a recovery rate of 97.7%. At the beginning of the test, a total recovery of 81.5% of the nominal concentration was measured in the test solution. At the end of the test (96 h), no test item was detected in the test solution with algae, and 32.3% was found in the stability sample without algae. It is assumed that by the end of the test most of the test item was probably either adsorbed or metabolised by the algal cells present in the test solution.


No significant inhibition of growth rate, biomass integral (A) and cell number, com­pared to the control, was observed. Therefore, based on nominal concentrations, a NOEC of ≥ 0.02 mg a.s./L was calculated for the test period 0-72 h and 0-96 h. No effect was found at the concentration tested (water solubility limit). Thus, it can be concluded that no algal toxicity is to be expected at the water saturation concentration of the test item.


Since the control growth validity criteria were met, the study is considered acceptable for the use in the risk assessment.


In the Draft Assessment Report for teflubezuron according Council Directive 91/414/EEC (2007) the 72 and 96-h EC50 of ≥ 0.02 mg a.s./L is indicated as key value for the toxicity of the test item to green algae. 


 


Supporting Information


The toxicity of the metabolites CL 902374 (CFPU, 3,5-dichloro-2,4-difluorophenyl­urea) and CL 9072373 (CFA, 3,5-dichloro-2,4-difluoroaniline) on the growth of green algae was investigated in 96-hour static laboratory studies on Pseudokirchneriella subcapitata.


For the supporting information no separate IUCLID endpoint study records have been prepared. For details please refer to the original Draft Assessment Report (DAR) Volume 3 Ecotoxicology and the EFSA conclusion for teflubenzuron according to Council Directive 91/414/EEC, Volume 3 – B.9 (AS) (2007).


Springborn Laboratories (1998): Effect of CL 902374 on growth of the freshwater algae Pseudokirchneriella subcapitata. Unpublished report, report  No. ECO 97-304, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference:  MCA 8.2.6/02


In the semi-static metabolite study of Springborn Laboratories (1998) according to OECD 201 (1984) green algae were exposed to a control, a solvent control and to 5 nominal CL 902374 (CFPU) concentrations of 0.16, 0.31, 0.63, 1.3, 2.5, 5.0, 7.5 and 10 mg CFPU/L. Therefore several stock solutions were prepared and diluted with OECD medium. Dimethylformamide (DMF) was used as solvent and was tested as solvent control at a concentration of 0.1 mL/L. The initial cell density was 104 cells/mL. Three sterile 250 mL-flasks were condi­tioned for each test concentration and the controls. The flasks were placed on an orbital shaker. The shaking rate was maintained at approximately 60 rpm. Based on the values for cell density, the NOEC for the area under the growth curve (AUC) and for the mean growth rate were determined for each test con­centration using one-way analysis of variance, William’s test or Dunnett’s test. Data were also checked for normality and for homogeneity. At test initiation, samples were taken for chemical analysis from each test con­centration and the control. At the end of the test, samples were taken from a composite of the three replicates of a concentration after isolation of the algae by centrifugation (20 minutes at 3,500 rpm). The samples were analysed by HPLC the same day they were taken.


The pH ranged from 7.64 to 7.84 at test initiation and from 8.00 to 9.14 at test termination. Conductivity ranged from 162 to 165 µSiemens/cm at hour 0 and from 145 to 164 µSiemens/cm at hour 72. Because of a technical problem dur­ing the first 24 hours of the definite test the temperature range was 21.3-24 °C (instead of 24 ± 1 °C). Between 24 and 72 hours the temperature range was 23.4 – 24.0 °C. The light intensity ranged from 6,000 – 7,700 lux. The conductiv­ity was inadvertently not recorded for the highest test concentration at test initia­tion and the light intensity was not recorded on test day 1 and 2. All deviations are not considered to have an impact on the results of the study.


The analytical verification showed a mean recovery of 87.5% of nominal (72 h). However, the results were calculated based on mean measured concentratons of 0. 0.14, 0.29, 0.58, 1.1, 2.5, 4.5, 7.0 and 9.7 mg CFPU/L. The study was deemed valid, since the increase in cell density in control culture within 27 h was 111.2*104 cells/mL, which corresponds to more than the minimum factor of 16. The cell density at concentrations higher or equal to 4.5 mg CFPU/L was significantly reduced compared to the solvent control. The NOEC (cell density) was 2.5 mg CFPU/L. The AUC (area under the growth curve) was significantly reduced com­pared to the solvent control at concentrations higher than or equal 7.0 mg CFPU/L. The NOEC (AUC) was determined to be 4.5 mg CFPU/L and an EC50 of 7.4 mg CFPU/L was determined based on mean measured concentrations. The growth rate was signifi­cantly reduced compared to the solvent control at concentrations higher or equal than 7.0 mg CFPU/L. The NOEC (growth rate) was determined to be 4.5 mg CFPU/L and an EC50 of > 9.7 mg CFPU/L was determined based on mean measured concentrations.


 


ECT (2003): A Study on the Toxicity of 3,5-dichloro-2,4-difluoroaniline to Algae (Pseudokirchneriella subcapitata). Unpublished report No. G4AG, according to Draft Assessment Report (2007) according to Council Directive 91/414/EEC, crossreference:  MCA 8.2.6/03


In the static metabolite study according to the EEC Directive 92/69 method C.3 (1992) green algae of Pseudokirchneriella subcapitata were exposed to control and 5 nominal CL 902373 (CFA) concentrations of 0.43, 0.94, 2.07, 4.55 and 10.00 mg CFA/L. A stock solution of CFA was prepared by dissolving 20.0 mg of the test item in 2 L algal medium (“Sammlung von Algenkulturen, Pflanzenphysiolo­gisches Institut der Unversität Göttingen (SAG) – List of Strains” (Schlösser 1982) with a pH of approximately 6.0). Then, the stock solution was stirred several times (in all for 80 min) and dispersed by ultrasonification (in all for 10 min) alternately. A liquid pre-culture was set up 3 days before the beginning of the test under test conditions. To start the test at day 0, the algal cells were transferred into the measuring flasks (104 cells/mL) which contain the test solutions, before the test solution was filled into the test vessels. The test was carried out in 300 mL Erlenmeyer flasks containing 100 mL test solution, at 23 ± 2°C with continuous illumination of 6,000 to 10,000 lx. The dilution water used was synthetic algal medium. There was no indication of interactions between this medium and the test substance. Four replicates were used for the control and the concentration levels and the exposure duration was 96 h. To verify the nominally applied concentrations, duplicate samples of the control, the highest, the lowest and one intermediate concentration were taken and stored deep-frozen until analysis.  Analysis was done using a HPLC/UV method. Probit analysis was used to calculate the EC50 values. One-way Analysis of Vari­ance (ANOVA), Cochran´s Test (testing of variance homogeneity) and subse­quent multi-comparison tests (Dunnett’s Test) were used to calculate whether there were significant differences between the growth of algae in the controls and the algae exposed to the various test item concentrations (ToxRat Profes­sional, version 2, 2003). In case of variance inhomogeneity, Bonferroni U-test after Holm was used instead of the Dunnett’s test procedure. For determination of the NOEC, the highest concentration level (1,000 mg CFA/L test item) was excluded from statistical analyses.


The pH value during the period of the test was 5.8/6.2 (min/max). The mean temperature was 23.81°C (min: 22.78°C, max: 24.99°C). In the analytical verification, the stock solution showed a recovery rate of 96.4% teflubenzuron. At the beginning of the test, an average recovery of 88.8% of the nominal concen­tration was measured in the test solution. At the end of the test (96 h), an aver­age recovery of 44.5% was measured in the test solution with algae, which is assumed to be due to adsorption of the test item to algal cells, and 82.3% was found in the stability sample without algae. The mean total recovery rate for all analysed test solutions was calculated as 62.6%. Since the mean total recovery rate in the test solutions was lower than 80% of the nominal concentrations, a correction of the results by a factor of 0.626 based on mean total recovery rate was performed.


The cell concentration in the control cultures increased by the factor 141, within three days (at least a factor of 16 is demanded by the guideline). Thus, the validity criterion was fulfilled. Based on mean measured concentrations the EbC50 value for biomass integral (AUC) was 1.26 mg CFA/L and the ErC50 for the growth rate was 2.99 mg CFA/L at 96 hours. The NOEC values for biomass integral (AUC) and growth rate at 96 hours were for both 0.27 mg CFA/L based on mean measured concentrations.


 


Conclusion


In the algal growth test with D. subspicatus according to EEC Directive 92/69 method C.3 (1992) the 96-hour EC50 and NOEC of the test item were determined to be of ≥ 0.02 mg a.s./L (nominal). Supporting information of toxicity studies on algal growth with the metabolites CL 902374 (CFPU) and CL 902373 (CFA) are available. In these studies with P. subcapitata the 96-hour EC50 values of CFU was > 9.7 mg CFPU/L (mean measured) and 2.99 mg CFA/L (mean measured). Based on the most reliable study on the active substance the 96-hour EC50 of the test item is considered to be ≥ 0.02 mg a.s./L. This value is fully in line with the conclusion drawn in the Draft Assessment Report for teflubenzuron prepared according to the Council Directive 91/414/EEC, Volume 3 – B.9 (AS), 2007.