diethyl pyridine-2,4-dicarboxylate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-Jun-2010 to 16-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.3: Algal Inhibi

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision 30-April-2008

Test material

Test material information

Constituent 1

Reference substance name:

Mexoryl SBU

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

Mexoryl SBU

Constituent 2

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Details on test material:

Name of test material: MEXORYL SBUThe test material is the same than the one mentioned in section 6.1.3

Sampling and analysis

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. In pre-experiments (non-GLP), the test item proved to be stable in the test water under these storage conditions.The concentrations of the test item MEXORYL SBU were determined in the duplicate test medium samples from all test concentrations. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.

Test solutions

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 50.02 mg of the test item completely in 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration 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

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.Nygaard et al. recommended describing the taxa within the Genus Raphidocelis HINDAK as:Raphidocelis subcapitata (KORSIKOV) nov. comb.Basionym:Ankistrodesmus subcapitatus KORSIKOVSyn.:Kirchneriella subcapitata KORSIKOVSyn.:Selenastrum capricornutum PRINTZSyn.:NIVA-CHL 1An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.The test species are recommended by the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72

Post exposure observation period:

not applicable

Test conditions

Hardness:

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

The water temperature during the test was maintained at 22 °C.

pH:

At the start of the test, the pH measured in the treatments was between 8.0 and 8.2. At the end of the test, pH values ranged between 8.5 and 9.1.

Dissolved oxygen:

not applicable

Salinity:

according to OECD test guideline

Nominal and measured concentrations:

The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control.At the start of the test, the measured concentrations of MEXORYL SBU in the test media of the nominal test concentrations of 1.0 to 100 mg/L were between 89 and 93% of the nominal values. Thus, the correct dosage of the test item at test start could be confirmed. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, 11 to 45% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test) were between 31 and 64% of the nominal values.

Details on test conditions:

Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. For further information please see section "Any other information on materials and methods incl. tables " below.50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7600 Lux (range: 6670 to 8040 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within ±15% from the average light intensity as recommended by the guideline.The selection of the test concentrations was based on the results of a range-finding test (non GLP). The following nominal concentrations of the test item were tested: 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item). The enlarged spacing factor of 3.2 between the test concentrations was chosen based on the results of the range-finding test, which documented a rather flat concentration-effect relationship. Therefore, a large concentration range had to be tested.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).A static test design was applied. The duration of the test was 72 hours.

Reference substance (positive control):

yes

Remarks:

tested twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

The measured concentrations of Mexoryl SBU in the test media of the nominal test concentrations of 1.0 to 100 mg/L were between 89 and 93% of the nominal values at the start of the test. Thus, the correct dosage of the test item at test start could be confirmed. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, 11 to 45% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test) were between 31 and 64% of the nominal values.Nominal test concentrationMean measured concentration of the test item(geometric mean)(mg/L)(mg/L)(% of nominal)1.00.31313.21.340104.4443215471006464The biological results were related to the mean measured test item concentrations.The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at all test concentrations (according to Williams t-tests, one-sided smaller, alpha = 0.05). After 72 hours of test duration, inhibitory effects ranged between1.8 and 6.5% for average growth rate and between 7.9 and 26% for yield. Inhibitory effects on yield were higher than on average growth rates. At the mean measured test item concentrations of 0.31 and 1.3 mg/L, yield was inhibited by 7.9 and 9.3% compared to the control, respectively, after 72 hours of test duration. The average growth rates were inhibited by 1.8 and 2.1%. According to Williams t-tests (one-sided smaller, alpha = 0.05) the inhibitory effects were statistically significant. However, the inhibitory effects (below 10% for yield) were not estimated as a biologically relevant toxic effect and the statistical significance was seen as a result of very low variability between treatment replicates. According to the guideline, concentrations provoking effects of 10 to 20% appear appropriate to represent the NOEC.Therefore, the overall 72-hour NOEC (for growth rate and yield) was determined to be 1.3 mg/L.The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.In the control the biomass increased by a factor of 98 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 (section-by-section growth rates) during 72 hours was 19%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.2%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.At the start of the test, the pH measured in the treatments was between 8.0 and 8.2. At the end of the test, pH values ranged between 8.5 and 9.1. The water temperature during the test was maintained at 22 °C.

Results with reference substance (positive control):

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 March 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.94 mg/L (study C86922), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 to 1.7 mg/L).

Reported statistics and error estimates:

The 72-hour EC10 and EC20 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis.The 72-hour EC50 values of the test item could not be calculated due to the low inhibitory effect of the test item on the algal growth at the tested concentrations. The 72-hour EC50 values were, therefore, directly determined from the raw data.For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test.

Any other information on results incl. tables

The measured concentrations of MEXORYL SBU in the test media of the nominal test concentrations of 1.0 to 100 mg/L were between 89 and 93% of the nominal values at the start of the test. Thus, the correct dosage of the test item at test start could be confirmed. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, 11 to 45% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test) were between 31 and 64% of the nominal values.

 

Nominal test concentration	Mean measured concentration of the test item (geometric mean)
(mg/L)	(mg/L)	(% of nominal)
1.0	0.31	31
3.2	1.3	40
10	4.4	44
32	15	47
100	64	64

The biological results were related to the mean measured test item concentrations.

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at all test concentrations (according to Williams t-tests, one-sided smaller,a = 0.05).

 

After 72 hours of test duration, inhibitory effects ranged between1.8 and 6.5% for average growth rate and between 7.9 and 26% for yield. Inhibitory effects on yield were higher than on average growth rates.

 

At the mean measured test item concentrations of 0.31 and 1.3 mg/L, yield was inhibited by 7.9 and 9.3% compared to the control, respectively, after 72 hours of test duration. The average growth rates were inhibited by 1.8 and 2.1%. According to Williams t-tests (one-sided smaller,a = 0.05) the inhibitory effects were statistically significant. However, the inhibitory effects (below 10% for yield)were not estimated as a biologically relevant toxic effect and the statistical significance was seen as a result of very low variability between treatment replicates. According to the guideline, concentrations provoking effects of 10 to 20% appear appropriate to represent the NOEC.

 

Therefore, the overall 72-hour NOEC (for growth rate and yield) was determined to be 1.3 mg/L.

The biological results can be summarized as follows (on the basis of mean measured concentrations of the test item):

 

 

Parameter	Growth rate	Yield
(0-72 h)
EC10  (mg/L)	>64	0.75
95% confidence interval	n.d.	0.41 – 1.2
EC20  (mg/L)	>64	14
95% confidence interval	n.d.	11 - 18
EC50  (mg/L)	>64	>64
95% confidence interval	n.d.	n.d.
NOEC (mg/L)	1.3	1.3
LOEC (mg/L)	4.4	4.4

 

n.d.   could not be determined

 

 

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

 

In the control the biomass increased by a factor of 98 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 (section-by-section growth rates) during 72 hours was 19%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.2%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

 

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

 

At the start of the test, the pH measured in the treatments was between 8.0 and 8.2. At the end of the test, pH values ranged between 8.5 and 9.1. The water temperature during the test was maintained at 22 °C.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at all test concentrations (according to Williams t-tests, one-sided smaller, alpha = 0.05). After 72 hours of test duration, inhibitory effects ranged between1.8 and 6.5% for average growth rate and between 7.9 and 26% for yield. Inhibitory effects on yield were higher than on average growth rates.

Executive summary:

The influence of the test item MEXORYL SBU on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3.

 

The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested in parallel with a control.

 

At the start of the test, the measured concentrations of MEXORYL SBU in the test media of the nominal test concentrations of 1.0 to 100 mg/L were between 89 and 93% of the nominal values. Thus, the correct dosage of the test item at test start could be confirmed. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, 11 to 45% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test) were between 31 and 64% of the nominal values.

 

Nominal test concentration (mg/L)	Mean measured concentration of the test item (geometric mean)
	(mg/L)	(% of nominal)
1.0	0.31	31
3.2	1.3	40
10	4.4	44
32	15	47
100	64	64

The biological test results (based on mean measured concentrations of the test item) were as follows:

Parameter (0-72 h)	Growth rate	Yield
EC10  (mg/L)	>64	0.75
95% confidence interval	n.d.	0.41 – 1.2
EC20  (mg/L)	>64	14
95% confidence interval	n.d.	11 - 18
EC50  (mg/L)	>64	>64
95% confidence interval	n.d.	n.d.
NOEC (mg/L)	1.3	1.3
LOEC (mg/L)	4.4	4.4

 

n.d. could not be determined