Environmental Project, 1041 – Pulse Effects of Herbicides on Periphyton in Streams and Recovery

Pulse Effects of Herbicides on Periphyton in Streams and Recovery

2 Materials and Methods

2.1 Achieving the natural populations of epiphytes
2.2 Exposure to herbicides
- 2.2.1 Effect of varying exposure time
- 2.2.2 Recovery experiment
2.3 Calculation of effect concentrations
2.4 Pigment analyses

2.1 Achieving the natural populations of epiphytes

Three frames each containing 170 round glass discs with a diameter of 1 cm were positioned in the mesothrophic and uncontaminated (due to almost no agricultural activity in the catchment area) stream Esrum Mølleå, located in the northern part of Sjælland, Denmark, in September 1999 and May 2000. After 2-3 weeks the glass discs were visibly coloured by colonising epiphytes growing on the glass surfaces and they were transported to the laboratory nearby. The glass discs were gently transferred to a white tray containing stream water and the discs were sorted with regard to density of attached epiphytes. Only glass discs, which were uniformly covered with epiphytes, were used in the experiments. Each disc selected was transferred to a glass vial containing 10 ml filtered stream water added 100 µM NaNO₃, 16 µM Na₂HPO₄, and 50 µM Na₂SiO₃·5H₂O, to ensure that nutrients were not limiting for the growth of the periphyton in the experiments.

2.2 Exposure to herbicides

In September 1999, the glass discs with epiphytes were exposed to pendimethalin, isoproturon, metribuzin, or hexazinone for 24 hours at concentrations 0, 0.4, 2, 10, and 50 µgl^-1 in triplicates. For isoproturon and metribuzin, additional concentrations of 250 and 1250 µgl^-1 were included in the experiments. Furthermore, for studying the effects of short-term exposure to pesticides, periphyton was also exposed to hexazinone, isoproturon and pendimethalin in the concentrations 0, 0.4, 2, 10, and 50 µgl^-1 in triplicates for 1 hour. The glass vials were incubated at in situ temperature (16 °C) and light intensity (250 µE m^-2s^-1) in an incubator in the laboratory. The effect of the pesticides on the periphyton was determined by measuring the photosynthetic activity by adding 1µCi ¹⁴C (The International Agency for ¹⁴C Determination, Denmark) to the vials by the end of the exposure period and incubating the vials for one hour as described above. Adding acetic acid until pH 2 stopped the incubations. The water was evaporated by drying the samples at 60 °C. The release of incorporated ¹⁴C-carbon was enhanced by addition of 1 ml concentrated dimetylsulfoxide. After half an hour 10 ml scintillation cocktail (UltimaGold, Packard) was added. The activity, as disintegration per minute (dpm), was calculated from the counts per minutes (cpm) data using an external standard technique and appropriate correction factors. The abiotic carbon fixation in all experiment <0.5% was estimated as the fixation of ¹⁴C in samples killed by addition of formalin to a final concentration to 1%.

2.2.1 Effect of varying exposure time

In May 2000 the effect of a varying exposure time (1- 48 h) was studied at different concentrations (0, 0.4, 2, 10, and 50 µgl^-1) of metribuzin. 270 vials each containing one glass disc with natural community of periphyton were prepared for the experiment. The periphyton were incubated with metribuzin in the different concentrations, and after 1, 2, 6, 12, 24, and 48 hours, the photosynthetic activity was determined in triplicates as described above. In order to determine the effect of metribuzin on the group composition of periphyton, 3 vials from each concentration were sampled from the experiment after 2, 24 and 48 hours. The 3 glass discs with periphyton from the same concentration were wrapped in a GF/F filter, immediately frozen in liquid nitrogen, and analysed within two months by HPLC as described below.

2.2.2 Recovery experiment

Vials were sampled 1, 2, 6, 12, 24, and 48 hours after the addition of Metribuzin for the recovery experiment. The Metribuzin containing stream water was decanted from each vial and immediately replaced by fresh filtered stream water with added nutrients. After 48 hours in fresh water the primary production was determined in triplicates as described above. Corresponding recovery of the group composition of the epiphytic communities exposed 2, 24, or 48 hours, was investigated after 48 hours in herbicide-free water.

2.3 Calculation of effect concentrations

No effect concentration (NEC) and effect concentrations (EC₅₀) were calculated for the photosynthetic activity measurements using log-linear interpolation as described in Petersen & Gustavson (1998). In some instances NEC could not be determined. Instead the lowest observed effect concentration (LOEC) was determined.

2.4 Pigment analyses

For HPLC analyses of the pigment composition the filter package with the 3 glass discs were thawed and placed in 6 ml 90 % acetone, sonicated on ice for 10 minutes and extracted for 24 hours at 4 °C. The filter and cell debris were filtered from the extract using disposable syringes and 0.2 µm Teflon syringe filters. 1ml extract and 0.3 ml water was transferred to HPLC vials and the vials were placed in the cooling rack of the HPLC. The samples were injected into a Shimadzu LC-10A HPLC system according to the method described by Wright et al. (1991), although the linear gradient was modified slightly: 0 min: 100 % A, 2 min: 100 % B, 2.6 min: 90 % B/10 % C, 13.6 min: 65 % B/35 % C, 20 min: 31 % B/69 % C, 28 min: 100 % B, 31 min: 100 % A. The HPLC system was calibrated with pigment standards from The International Agency for ¹⁴C Determination, DHI – Water and Environment, Denmark. Peak identities were routinely confirmed by diode array.