Dodecan-1-ol, ethoxylated

Endpoint summary

Description of key information

Short term toxicity of Fish:

The short term toxicty to fish was studied on fresh water fish danio rerio by following OECD 203 test guidelines. Juvenile fishes, of average size 1.46 cm with loading rate of 0.1193 g/l was taken and exposed to the test chemical for the period of 96 hours and mortality was observed at regular intervals of 2±0.5 h, 5 ± 1 h, 24 ± 2, 30 ± 2, 48 ± 2, 54 ± 2, 72 ± 2, 78 ± 2 and 96 ± 2 h. Before initiation of study, test organisms were acclimitised to experimental conditions for the period of at least for 9 days (including 7 days acclimatization) before using it for test, no mortality was observed during the acclimatization period. The test medium was reconstituted water, as described in the OECD test guideline. The conductivity of the deionised water used in the preparation of the test medium was 0.054 µS/cm during range finding and definitive test. The test medium was aerated until the dissolved oxygen concentration equated the air-saturation value. The test medium so prepared was set aside without further aeration for a minimum period of at least two days before use and used within 7 days after preparation for the treatment. The range finding test was carried outas a non-GLP testwith test concentrations of0.001, 0.01, 0.1, 1, 10 and 100mg/L of the test medium along with a negative control in a semi-static mode (renewal of test solution at every 24 h interval). Five fish were used for each test solution with single replicate per test concentration. Based on the outcomes the definitive test was carried out using nominal test concentrations0.5, 1.0, 2.0, 4.0, 8.0 and 16mg/L along with control in a static mode. Ten fish were used for each test solution with single replicate per test concentration. To prepare test concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L,10, 20, 40, 80, 160 and 320 mg test item was added to test aquarium containing 20 L of test medium separately.For negative control test aquarium containing 20 L of test medium was used.Test vessels were randomly positioned in the test area and shielded from unwanted disturbance. The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 106.85 to 118.90 %(RSD was 2.55 to 8.48 %)at the start and 98.13 to 106.47 %(RSD was 1.75 to 9.59 %)at the end of the test (96 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%). In the range finding test, there was no mortality of fish in the negative control and at the tested concentrations of 0.001, 0.01 and 0.1 mg/L up to 96 h. There was 20, 100 and100 %mortality of fish at the tested concentrations of 1, 10 and 100 mg/L at 96 h exposure, respectively. In the definitive test, fish starved at least for 24 h were exposed to test chemical at nominal concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L along with a negative control under static mode. Fish were observed for toxic signs and mortality for 96 h. Each group had single replicate with ten fish. There was no mortality of fish in the negative control and at the tested concentration of 0.5 mg/L at 96 h exposure.There was 0, 40, 50, 60 and 80 % mortality at 24 hours; 0, 40, 50, 70 and 90 % mortality at 48 hours; and 10, 50, 70, 90 and 100 % mortality at 72 and 96 hours at the tested concentrations of1.0, 2.0, 4.0, 8.0 and 16.0 mg/L.On the basis of observation made during the test, the NOEC (No Observed Effect Concentration) was 0.5 mg/L based on nominal concentrations. On the basis of observation made during the test, the LOEC (Low Observed Effect Concentration) was 1.0 mg/L based on nominal concentrations. The LC50 value for test chemical at 24 hours was 3.76 mg/L based on nominal concentrations. The LC50 value for test chemical at 48 hours was 3.354 mg/L with fiducial limits at 95% ranging from 0.396 to 6.226 based on nominal concentrations. The LC50 value for test chemical at 72 and 96 hours was 2.427 mg/L with fiducial limits at 95% ranging from 1.6769 to 3.5123 based on nominal concentrations. As the test chemical is readily biodegradable, it cannot be classified as per CLP classification criteria.

Short term toxicity Daphnia:

The acute immobilization effect of the test item was studied onDaphnia magnafor 48 hours. the study was conducted by following the OECD 202 test guidelines, by observing the immobilisation caused during exposure period of 24 and 48 hours. Test system was daphnia magna with age less than 24 hours. During acclimitisation, test medium in which the parent daphnia were maintained was changed at least twice a week and they were fed with the unicellular green alga,Chlorella vulgaris. The medium used for the test was reconstituted water as described in the OECD test guideline 202 (OECD, 2004). The conductivity of the deionized water used for the preparation of the test medium was 0.054 µS/cm. The hardness and total organic carbon (TOC) of reconstituted water was 250 CaCO₃/L and 0.8 mg/L. The test medium so prepared was set aside without aeration for a minimum period of 2 days before use and was used for the treatment within 2-6 days of preparation. One day prior to the start of the treatment, about 20 adults were incubated in approximately 2 L of reconstituted water. The following day, the young ones hatched which were less than 24 hour old were collected and used in the tests. During range finding study test organism was taken and exposed to the concentrations ranging form 0.01, 0.1,1, 10 and 100 mg/L along with control. There was no Immobilization of daphnia observed at the tested concentrations of 0.01, 0.1 and 1 at 24 hours exposure and there was no Immobilization of daphnia observed at the tested concentrations of 0.01 and 0.1 at 48 hours exposure.however, There was 30 and 60 % immobility of the daphnia observed at 24 hour at the tested concentrations of 10 and 100 mg/L, respectively and 10, 70 and 90 % immobility of the daphnia observed at 48 hour at the tested concentrations of 1, 10 and 100 mg/L, respectively. Based on the outcomes In definitive test,Daphnia magnaless than 24 hours old were exposed to the nominal concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L. The number of daphnia immobilized was recorded at 24 and 48 hours exposure. There was no immobilization of daphnia in the negative control and at the tested concentrations of 0.5 and 1.0 mg/L at 24 and 48 hours of exposure. The immobilization of daphniawas 5, 10, 15 and 75 % at 24 h and 10, 15, 20 and 90 % at 48 h exposure at 2.0, 4.0, 8.0 and 16 mg/L, respectively. The stability test results concluded that the test item was stable in the test medium at 48 h at nominal concentrations.The active ingredient concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 94.31 to 105.59 %(RSD was 2.52 to 7.97%)at the start and 93.98 to 116.77 %(RSD was 3.48 to 19.83 %)at the end of the test (48 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%). All the validity criteria was maintained within the acceptibility domain, such as, there was no mortality in test controls and the dissolved oxygen concentration was > 3mg/l. To validate the study reference study was also conducted using potassium dichromate for 48 hours and EC50 was recorded for 24 and 48 hours.

The EC50 value for test chemical at 24 hours and 48 hours was 11.9099 mg/L and 9.1313 mg/L with fiducial limits at 95 per cent ranging from 8.55403 to 16.58231 and

2.76515 to 14.3770 respectively based on nominal concentrations. On the basis of observation made during the test, the LOEC (Low Observed Effect Concentration) was 2.0 mg/L based on nominal concentrations. On the basis of observation made during the test, the NOEC (No Observed Effect Concentration) was 1.0 mg/L based on nominal concentrations.  

Short term toxicity to Algae;

The test chemical was studied for its effects on fresh water algae P. Subcapitata by following OECD 201 test guidelines. The test organism was obtained from ATCC (American type culture collection) 22662, which was recommended as per guideliners. Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 24°C (±2°C) under a white fluorescent continuous illumination of 8000 ± 1600 Lux.  Test medium was algal medium as described in the OECD test guideline 201 (OECD 2006) The test medium was prepared by mixing 100 mL stock solution 1 and 10 mL each stock solution 2, 3 and 4 with 5 L of sterilized water and then making up the volume to 10 L using sterilized water. The pH of the medium was 7.68 during definitive test. To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculatingRaphidocelis subcapitataof continuous/mother culture to test medium 3 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions. The cell density of inoculum culture was 217.25 x 10⁴/mL during range finding and 231 x 10⁴/mL during definitive test. All test conditions during range finding and definitive test was similar such as exposure period (72h), test containers (250 ml conical flask), pH(7.64 -7.74), temperature (22.2 -22.6°C), continues light intensity (6963 -7100 lux) shaking of incubator (150 rpm). Range finding test was conducted to select the test concentrations for the definitive test with a negative control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L, each single replicateTest item exhibited 0, 0, 10.35, 83.81 and 100 % Inhibition at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h, respectively. based on the results obtained theDefinitive test was carried out using 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L (factor of 2.0) along with a negative control. At test concentration of the test concentrationsof0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L, the observed inhibition in the percent growth rate of algal biomass was 0, 7.84, 47.71, 67.47, 86.23 and 96.42 %; and the percent yield inhibition was 0, 29.49, 88.79, 96.02, 98.95 and 99.79 %, respectively when compared to that of the negative control during the test period at 72 h

The end point values for growth rate and yield 72 hours are presented below based on nominal concentration.

 

At 72 hours	EC values in mg/L (fiducial limits)
ErC50	2.737 (1.761-3.9537)
ErC20	1.255 (0.80749-1.81297)
ErC10	0.8349 (0.53718-1.20606)
EyC50	1.225 (0.2544-1.9892)
EyC20	0.6721 (0.13957-1.0913)
EyC10	0.491 (0.10198-0.79734)
LOEC	1.0
NOEC	0.5

The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 103.47 to 117.02 %(RSD was 2.27 to 5.88 %)at the start and 93.03 to 114.59 %(RSD was 0.29 to 4.69 %)at the end of the test (72 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%).No measurable concentration of test item was recorded in the negative control samples.

Based on outcomes on median effective concentration, test chemical cannot be classified as per CLP classification criteria, since chemical is readily biodegradable

Additional information

Short Term toxicity to fish

The short term toxicty to fish was studied on fresh water fish danio rerio by following OECD 203 test guidelines. Juvenile fishes, of average size 1.46 cm with loading rate of 0.1193 g/l was taken and exposed to the test chemical for the period of 96 hours and mortality was observed at regular intervals of 2±0.5 h, 5 ± 1 h, 24 ± 2, 30 ± 2, 48 ± 2, 54 ± 2, 72 ± 2, 78 ± 2 and 96 ± 2 h. Before initiation of study, test organisms were acclimitised to experimental conditions for the period of at least for 9 days (including 7 days acclimatization) before using it for test, no mortality was observed during the acclimatization period. The test medium was reconstituted water, as described in the OECD test guideline. The conductivity of the deionised water used in the preparation of the test medium was 0.054 µS/cm during range finding and definitive test. The test medium was aerated until the dissolved oxygen concentration equated the air-saturation value. The test medium so prepared was set aside without further aeration for a minimum period of at least two days before use and used within 7 days after preparation for the treatment. The range finding test was carried outas a non-GLP testwith test concentrations of0.001, 0.01, 0.1, 1, 10 and 100mg/L of the test medium along with a negative control in a semi-static mode (renewal of test solution at every 24 h interval). Five fish were used for each test solution with single replicate per test concentration. Based on the outcomes the definitive test was carried out using nominal test concentrations0.5, 1.0, 2.0, 4.0, 8.0 and 16mg/L along with control in a static mode. Ten fish were used for each test solution with single replicate per test concentration. To prepare test concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L,10, 20, 40, 80, 160 and 320 mg test item was added to test aquarium containing 20 L of test medium separately.For negative control test aquarium containing 20 L of test medium was used.Test vessels were randomly positioned in the test area and shielded from unwanted disturbance. The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 106.85 to 118.90 %(RSD was 2.55 to 8.48 %)at the start and 98.13 to 106.47 %(RSD was 1.75 to 9.59 %)at the end of the test (96 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%). In the range finding test, there was no mortality of fish in the negative control and at the tested concentrations of 0.001, 0.01 and 0.1 mg/L up to 96 h. There was 20, 100 and100 %mortality of fish at the tested concentrations of 1, 10 and 100 mg/L at 96 h exposure, respectively. In the definitive test, fish starved at least for 24 h were exposed to test chemical at nominal concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L along with a negative control under static mode. Fish were observed for toxic signs and mortality for 96 h. Each group had single replicate with ten fish. There was no mortality of fish in the negative control and at the tested concentration of 0.5 mg/L at 96 h exposure.There was 0, 40, 50, 60 and 80 % mortality at 24 hours; 0, 40, 50, 70 and 90 % mortality at 48 hours; and 10, 50, 70, 90 and 100 % mortality at 72 and 96 hours at the tested concentrations of1.0, 2.0, 4.0, 8.0 and 16.0 mg/L.On the basis of observation made during the test, the NOEC (No Observed Effect Concentration) was 0.5 mg/L based on nominal concentrations. On the basis of observation made during the test, the LOEC (Low Observed Effect Concentration) was 1.0 mg/L based on nominal concentrations. The LC50 value for test chemical at 24 hours was 3.76 mg/L based on nominal concentrations. The LC50 value for test chemical at 48 hours was 3.354 mg/L with fiducial limits at 95% ranging from 0.396 to 6.226 based on nominal concentrations. The LC50 value for test chemical at 72 and 96 hours was 2.427 mg/L with fiducial limits at 95% ranging from 1.6769 to 3.5123 based on nominal concentrations. As the test chemical is readily biodegradable, it cannot be classified as per CLP classification criteria.

In an experimental study from peer reviewed journal (Mikio KiKuchi et. al., 1984), an acute toxicity test was conducted for 48 hrs for assessing the effect of test chemical on Oryzias latipes. The test was performed in accordance to the Japanese Industrial Standard (JIS) K0102 (1981) Testing methods for Industrial Wastewater. Adult Oryzias latipes (Japanese medaka) (details regarding average weight and length has been provided in table 2) obtained from a commercial fish farmer in Tokyo was used as a test organism for the study. Test fishes were held in the laboratory for a period of 2 -4 weeks at 20 -23°C prior to use and were fed daily with excess of dry commercial diet pellet. Exact test chemical conc. was not reported, but test concentrations were taken which was increased by geometrical progression of 1.4. Test fishes (total no. of test fishes varies for 4 different runs as mentioned in table 2) were exposed to test chemical in a 3 -5 l glass beaker. The mass/volume ratio was never exceeded of about 1 g fish per liter of water per day. Fishes were not fed during the study. Test solution was renewed after 24 hr. The test vessels were kept in a constant temperature water bath of 21 to 22°C, pH6.7 to 7.1, dissolved oxygen ranges from 8.5 mg/l (at the start of test) to 5 mg/l (at the end of test), hardness of water 25 mg of CaCO3, respectively. Aeration was not provided in test vessels. All tests were carried out in 1 replicate on 4 different runs. Mouth and opercular movement were used to determine the life or death of the test organism. The LC50 were estimated by graphical interpolation of observed survivals greater and less than 50%.On the basis of mortality of the test organism Oryzias latipes (Japanese medaka), the 48 hr LC50 was determined to be 2.4 mg/l.

 

Another short term fish toxicity was conducted for 48 hrs for assessing the effect of test chemical (authoritative database, 2018 and secondary source, 2019). Study was performed using Cyprinus carpio (Commn carp) (larvae) in a static system at 20°C. Test chemical concentration was not verified analytically. On the basis of effect on mortality of the test organism, the 48 hr LC50 was determined to be 3.72 mg/l (95% C. I. – 3.11 to 4.47 mg/l) (nominal concentration).

 

In a supporting weight of evidence study, short term toxicity to fish was conducted for 48 hrs for assessing the effect of test chemical (authoritative database, 2018 and secondary source, 2019). Poecilia reticulata (guppy) was used as a test organism. Study was performed in a static system at 20°C. Test chemical concentration was not verified analytically. On the basis of effect on mortality of the test organism, the 48 hr LC50 was determined to be 8.61 mg/l (95% C. I. – 7.14 to 10.33 mg/l) (nominal concentration).

 

The overall results for the target chemical suggest that, the chemical has tendency to pose toxic effects to freshwater fish in lower concentrations. However, test chemical is readily biodegradable in nature, hence it cannot be classified in to hazardous category, based on CLP classification criteria.

Short term toxicity to Aquatic invertebrate:

The acute immobilization effect of the test item was studied onDaphnia magnafor 48 hours. the study was conducted by following the OECD 202 test guidelines, by observing the immobilisation caused during exposure period of 24 and 48 hours. Test system was daphnia magna with age less than 24 hours. During acclimitisation, test medium in which the parent daphnia were maintained was changed at least twice a week and they were fed with the unicellular green alga,Chlorella vulgaris. The medium used for the test was reconstituted water as described in the OECD test guideline 202 (OECD, 2004). The conductivity of the deionized water used for the preparation of the test medium was 0.054 µS/cm. The hardness and total organic carbon (TOC) of reconstituted water was 250 CaCO₃/L and 0.8 mg/L. The test medium so prepared was set aside without aeration for a minimum period of 2 days before use and was used for the treatment within 2-6 days of preparation. One day prior to the start of the treatment, about 20 adults were incubated in approximately 2 L of reconstituted water. The following day, the young ones hatched which were less than 24 hour old were collected and used in the tests. During range finding study test organism was taken and exposed to the concentrations ranging form 0.01, 0.1,1, 10 and 100 mg/L along with control. There was no Immobilization of daphnia observed at the tested concentrations of 0.01, 0.1 and 1 at 24 hours exposure and there was no Immobilization of daphnia observed at the tested concentrations of 0.01 and 0.1 at 48 hours exposure.however, There was 30 and 60 % immobility of the daphnia observed at 24 hour at the tested concentrations of 10 and 100 mg/L, respectively and 10, 70 and 90 % immobility of the daphnia observed at 48 hour at the tested concentrations of 1, 10 and 100 mg/L, respectively. Based on the outcomes In definitive test,Daphnia magnaless than 24 hours old were exposed to the nominal concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L. The number of daphnia immobilized was recorded at 24 and 48 hours exposure. There was no immobilization of daphnia in the negative control and at the tested concentrations of 0.5 and 1.0 mg/L at 24 and 48 hours of exposure. The immobilization of daphniawas 5, 10, 15 and 75 % at 24 h and 10, 15, 20 and 90 % at 48 h exposure at 2.0, 4.0, 8.0 and 16 mg/L, respectively. The stability test results concluded that the test item was stable in the test medium at 48 h at nominal concentrations.The active ingredient concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 94.31 to 105.59 %(RSD was 2.52 to 7.97%)at the start and 93.98 to 116.77 %(RSD was 3.48 to 19.83 %)at the end of the test (48 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%). All the validity criteria was maintained within the acceptibility domain, such as, there was no mortality in test controls and the dissolved oxygen concentration was > 3mg/l. To validate the study reference study was also conducted using potassium dichromate for 48 hours and EC50 was recorded for 24 and 48 hours.

The EC50 value for test chemical at 24 hours and 48 hours was 11.9099 mg/L and 9.1313 mg/L with fiducial limits at 95 per cent ranging from 8.55403 to 16.58231 and

2.76515 to 14.3770 respectively based on nominal concentrations. On the basis of observation made during the test, the LOEC (Low Observed Effect Concentration) was 2.0 mg/L based on nominal concentrations. On the basis of observation made during the test, the NOEC (No Observed Effect Concentration) was 1.0 mg/L based on nominal concentrations.                                    

In an experimental study (Study report, 2019), an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp., Acute Immobilization Test”. The stock solution was prepared by dissolving 100mg of test chemical in 1L of ADaM’s media, From which further test concentrations of 0, 1, 2, 4, 8 and 16 mg/L were prepared. The highest and lowest exposed test chemical concentrations were verified analytically at day 0 and day 2 which has been satisfactorily maintained within ± 20 % of the nominal initial concentration throughout the test. Study was performed using 10 daphnids in a static fresh water system. Total 10 Daphnids were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20±2°C, pH 7.3, hardness of water 150.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. One control containing solubilizing agent was also run simultaneously during the study. The animals were exposed to medium (i.e.a beaker containing only medium) and the tested chemical during 48 hour. The daphnids were moving slowly as compared to control. On the basis of this, the median effect concentration (EC50 (48 h)) value was determined to be 5.2 mg/L (nominal concentration). Thus, based on the EC50 value, test chemical can be considered as toxic to aquatic invertebrates and can be expected to be classified in ‘Aq. chronic category 2’. However, test chemical is readily biodegradable in water, chemical is considered as non-toxic to aquatic invertebrates and thus, considered to be ‘not classified’ as per the CLP classification criteria.

 

In a supporting weight of evidence study, short term toxicity to aq. Invertebrate study was conducted for 96 hrs for assessing the effect of test chemical (C. Pantani et. al., 1997 and secondary source, 2019). The study was performed under static conditions at 8±0.5°C. Gammarus italicus and Echinogammarus tibaldii collected near the spring river Vera (L’Aquila, Italy) were used as a test organism. Male test organisms were acclimatized for about 3 days in cool reconstituted water, which received artificial oxygenation. During the acclimation period, test organism was fed with dry poplar leaves previously soaked in spring water in order to enrich them with fungi and bacteria. Feeding was stop 24 hrs before the experiment. The reconstituted water has hardness 240 mg/l as CaCO3, alkalinity 55 mg/l as CaCO3 and pH 7.9±0.5, respectively. Six concentration of test chemical was taken based on the range finding study with the same logarithmic interval. Study was performed in 1 l glass jar containing 250 ml of the precooled aeration solution for 96 hrs. No aeration was provided in the test vessels. Test organisms was not fed during the test. All tests (test vessels including the control) were performed in 2 replicates using 20 test organisms of Echinogammarus tibaldii and less no. of Gammarus italicusi / replicate. Test organisms which showed no movements of the pleopodes when gently prodded with a spatula after 96 hr were considered as dead. Data was analysed through probits and the LC50 was expressed as nominal concentrations. On the basis of effect on mobility of the test organism G. italicus and E. tibaldii, the 96 hr LC50 was determined to be 7.6 mg/l (95% C. I. – 7.3 to 8.1 mg/l) and 3.6 mg/l (3.4 to 3.8 mg/l) (nominal concentration), respectively. Thus, test chemical can be considered as toxic to aquatic invertebrates. Since the test chemical is readily biodegradable in water, chemical is considered to be non-toxic to aquatic invertebrates.

 

For the test chemical, short term toxicity to aq. Invertebrate study was conducted for 24 hrs for assessing the effect of test chemical (C. Pantani et. al., 1990). The study was performed under static conditions. Gammarus italicus collected near the spring river Vera (L’Aquila, Italy) was used as a test organism. Male test organisms were acclimatized for about 3 days in artificial water at a constant temperature of 8°C, which received artificial oxygenation. During the acclimation period, test organism was fed with dry poplar leaves previously soaked in spring water in order to enrich them with fungi and bacteria. Feeding was stop 24 hrs before the experiment. Oxygenation was not provided in the test vessels. In addition to this, test organism was not fed during the study. Test fishes were exposed to the test chemical concentration at 8°C under test conditions which includes hardness 240 mg/l as CaCO3, alkalinity 55 mg/l as CaCO3 and pH 8.1, respectively. Test organisms which showed no movements of the pleopodes when gently prodded with a spatula after 24 hr were considered as dead. Data was analysed through probit with the help of a program in BASIC and the LC50 was expressed in mg/l. On the basis of effect on mobility of the test organism Gammarus italicus,the 48 hr LC50 was determined to be 9.45 mg/l (95% C. I. – 8.83 to 10.18 mg/l) (nominal concentration).Thus, test chemical can be considered as toxic to aquatic invertebrates. Since the test chemical is readily biodegradable in water, chemical is considered as non-toxic to aquatic invertebrates.

 

Another short term toxicity to aq. Invertebrate study was conducted for 48 hrs for assessing the effect of test chemical (authoritative database, 2018 and secondary source, 2019). This test was performed using Daphnia magna (water flea) under static conditions at 20°C.No analytical monitoring was performed of exposed test chemical concentrations. On the basis of effect on themortality of the test organism, the 48 hr LC50 value was determined to be 6.46 mg/l (95% C. I. – 4.78 to 7.58 mg/l) (nominal concentration). Based on this value, test chemical can be considered as toxic to aquatic invertebrates. Since the test chemical is readily biodegradable in water, chemical is non-toxic to aquatic invertebrates and is considered to be ‘not classified’ as per the CLP classification criteria.

 

On the basis of the above results, it can be concluded that the test chemical can be considered as toxic to aquatic invertebrates. However, test chemical is readily biodegradable in water, chemical is considered as non-toxic to aquatic invertebrates and thus, considered to be ‘not classified’ as per the CLP classification criteria.

Short term toxicity to Algae;

The test chemical was studied for its effects on fresh water algae P. Subcapitata by following OECD 201 test guidelines. The test organism was obtained from ATCC (American type culture collection) 22662, which was recommended as per guideliners. Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 24°C (±2°C) under a white fluorescent continuous illumination of 8000 ± 1600 Lux.  Test medium was algal medium as described in the OECD test guideline 201 (OECD 2006) The test medium was prepared by mixing 100 mL stock solution 1 and 10 mL each stock solution 2, 3 and 4 with 5 L of sterilized water and then making up the volume to 10 L using sterilized water. The pH of the medium was 7.68 during definitive test. To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculatingRaphidocelis subcapitataof continuous/mother culture to test medium 3 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions. The cell density of inoculum culture was 217.25 x 10⁴/mL during range finding and 231 x 10⁴/mL during definitive test. All test conditions during range finding and definitive test was similar such as exposure period (72h), test containers (250 ml conical flask), pH(7.64 -7.74), temperature (22.2 -22.6°C), continues light intensity (6963 -7100 lux) shaking of incubator (150 rpm). Range finding test was conducted to select the test concentrations for the definitive test with a negative control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L, each single replicateTest item exhibited 0, 0, 10.35, 83.81 and 100 % Inhibition at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h, respectively. based on the results obtained theDefinitive test was carried out using 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L (factor of 2.0) along with a negative control. At test concentration of the test concentrationsof0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L, the observed inhibition in the percent growth rate of algal biomass was 0, 7.84, 47.71, 67.47, 86.23 and 96.42 %; and the percent yield inhibition was 0, 29.49, 88.79, 96.02, 98.95 and 99.79 %, respectively when compared to that of the negative control during the test period at 72 h

The end point values for growth rate and yield 72 hours are presented below based on nominal concentration.

 

At 72 hours	EC values in mg/L (fiducial limits)
ErC50	2.737 (1.761-3.9537)
ErC20	1.255 (0.80749-1.81297)
ErC10	0.8349 (0.53718-1.20606)
EyC50	1.225 (0.2544-1.9892)
EyC20	0.6721 (0.13957-1.0913)
EyC10	0.491 (0.10198-0.79734)
LOEC	1.0
NOEC	0.5

The concentration analysis in all test concentrations showed that the mean percent recovery with nominal concentration was in the range of 103.47 to 117.02 %(RSD was 2.27 to 5.88 %)at the start and 93.03 to 114.59 %(RSD was 0.29 to 4.69 %)at the end of the test (72 hour) indicating that the results were within the acceptable limit (80 to 120 % of the nominal concentration with an RSD of<20%).No measurable concentration of test item was recorded in the negative control samples.

Based on outcomes on median effective concentration, test chemical cannot be classified as per CLP classification criteria, since chemical is readily biodegradable

 

In an experimental key study from peer reviewed journal (T. Wind et. al., 2006), an algal growth inhibition test was conducted for 72 hrs for assessing the growth inhibition potentialof test chemical on green algae. This test was performed by following the OECD Guideline 201 (Alga, Growth Inhibition Test) (1984) and EC Directive 92/69/EWG under static condition. Scenedesmus subspicatus (green algae) obtained from the Department of Plant Physiology (University of Goettingen/Germany) was used as a test organism. Initial cell density of the culture used was 10000cells/ml. The algae was cultured in mineral salts media according to the Directive 92/69/EWG, Annex C3 which corresponds to OECD 201 (1984). On the basis of range finding studies, five to seven different test chemical concentrations (nominal) were taken for the toxicity the study. Dilution series were constructed using factors between 2 to 3 depending on the substance and test. Analytical monitoring of test chemical conc. was carried out by GC analysis. Standard OECD test medium was used for the study. Axenic cultures of green algae were exposed to various nominal concentration of test chemical in 500 ml flask containing algal suspension. Test vessel were placed in a Clim-o-Shake® (horizontal shaker) for 72 hrs at a room at a temperature of 23 ± 2°C with a continuous uniform illumination of 2000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 100 revolutions per minute throughout the study period. Control was also included in the test. All the tests were performed in triplicates. Cell density was calculated by Calibrated Coulter Counter after every 24 hr. Statistical analysis was carried out by exposure-response modeling (point estimated by regression) and threshold estimation (analysis of variance) techniques. Exposure-response modeling employed the non-linear, iterative regression technique. It has the advantage of estimating the reliable ECx values where x signifies a specified effect level between 1 and 99 while including the common non-monotonic trends observed at low exposure concentrations in algal test (such as the hormetic response). Thresholds for the NOEC and LOEC were evaluated based on the one-way analysis of variance (ANNOVA) followed by an a posteriori Dunnet’s multiple range test. All statistical analyses were conducted in SAS. On the basis of effect on growth rate and AUG of the test organism Scenedesmus subspicatus, the 72 hr EC50 value was determined to be 0.2826 mg/l (EC50 = 0.00103 mM (95% C. I. 0.00645 to 0.001653 mM)) and 0.179 mg/l (EC50 = 0.00653 mM (95% C. I. 0.000369 to 0.00167 mM)), respectively. As test concentrations are maintained within 20% of the measured initial concentrations throughout the test, the median effect concentrations (EC50) reported was considered as nominal concentrations.  Thus, based on the ErC50 value (0.2826 mg/l), test chemical can be considered as toxic to aq. algae and thus can be considered to be classified in ‘Aquatic acute 1 category’ as per CLP classification criteria.

 

In a supporting study, a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata (Study report, 2019). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Pseudokirchneriella subcapitata obtained from Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium. Initial cell density of the culture was kept at 10000 cells/ml. The test solution was prepared by dissolving 300 mg of test chemical in 300 ml of OECD Media This stock solution was kept for stirring for 48 hours to obtain a homogenous solution for the experiment. The highest and lowest concentration of exposed test chemical was verified analytically at day 0 and day 3, which has been satisfactorily maintained within ± 20 % of the nominal initial concentration throughout the test. Green algae were exposed to nominal concentration of test chemical (0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L, 8 mg/L, and 16 mg/L, respectively) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control was also run simultaneously during the study. The cultures were counted and observed daily under microscope using heamocytometer to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increased exponentially by a factor of at least 16 within the 72 hr test period which corresponds to a specific growth rate of 0.92 per day, the mean coefficient of variation by section specific growth rate in the control cultures did not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures did not exceeded 7%, thus, fulfilling the validity criteria. In control vessel, all cells appeared healthy, sickle shape and green throughout the study duration. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs EC50 value was determined to be 0.82 mg/l. Thus, based on the EC50 value, chemical is considered as toxic to aquatic algae and thus considered to be classified in 'Aquatic acute 1 category' as per CLP classification criteria.

 

Another toxicity to aquatic algae and cyanobacteria study (Secondary source, 2009) was conducted for 72 hrs for assessing the effect of test chemical on Scenedesmus subspicatus (green algae). The study was performed according to the EC Directive 92/69/EWG in a static system. On the basis of the effect on growth rate and biomass of the test organism, the 72 hrs NOEC, ErC50 and EbC50 value was determined to be 0.09 mg/l, 0.43 mg/l and 0.18 mg/l, respectively. Thus, based on the ErC50 value (0.43mg/l), test chemical can be considered as toxic to aq. algae and thus can be considered to be classified in ‘Aquatic acute 1 category’ as per CLP classification criteria.

 

For the test chemical, toxicity to aquatic algae and cyanobacteria study was conducted for 8 days for assessing the effect of test chemical (HSDB, 2018 and secondary source, 2019). The study was performed in a static system using Scenedesmus quadricauda as a test organism at 20°C. Test chemical concentrations was not verified analytically. On the basis of the effect on Chlorophyll measurement of the test organism, the 8 days EC50 value was determined to be 3.3 mg/l. Thus, test chemical can be expected to be toxic to aquatic algae.

 

On the basis of the above results, Since the test chemical is readily biodegradable in water, thus it cannot be classified as per CLP classification criteria.